Optimized multi-epitope constructs and uses thereof

ABSTRACT

The invention relates to the field of biology. In particular, it relates to multi-epitope nucleic acid and peptide vaccines and methods of designing such vaccines to provide increased immunogenicity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application60/415,463 filed Oct. 3, 2002, and to U.S. Provisional Application60/419,973, filed Oct. 22, 2002, which are herein incorporated byreference.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

Part of the work performed during development of this invention utilizedU.S. Government funds. The U.S. Government has certain rights in thisinvention.

FIELD OF THE INVENTION

This present invention relates to the field of biology. In particular,it relates to multi-epitope nucleic acid vaccines and methods ofdesigning such vaccines to provide increased immunogenicity.

BACKGROUND

The technology relevant to multi-epitope (“minigene” e.g., “epigene”vaccines is developing. Several independent studies have establishedthat induction of simultaneous immune responses against multipleepitopes can be achieved. For example, responses against a large numberof T cell specificities can be induced and detected. In naturalsituations, Doolan et al (Immunity, Vol. 7(1):97-112 (1997))simultaneously detected recall T cell responses, against as many as 17different P. falciparum epitopes using PBMC from a single donor.Similarly, Bertoni and colleagues (J Clin Invest, Vol. 100(3):503-13(1997)) detected simultaneous CTL responses against 12 differentHBV-derived epitopes in a single donor. In terms of immunization withmulti-epitope nucleic acid vaccines, several examples have been reportedwhere multiple T cell responses were induced. For example, minigenevaccines composed of approximately ten MHC Class I epitopes in which allepitopes were immunogenic and/or antigenic have been reported.Specifically, minigene vaccines composed of 9 EBV (Thomson et al., ProcNatl Acad Sci USA, Vol. 92(13):5845-9 (1995)), 7 HIV (Woodberry et al.,J Virol, Vol. 73(7):5320-5 (1999)), 10 murine (Thomson et al., JImmunol, Vol. 160(4):1717-23 (1998)) and 10 tumor-derived (Mateo et al.,J Immunol, Vol. 163(7):4058-63 (1999)) epitopes have been shown to beactive. It has also been shown that a multi-epitope DNA plasmid encodingnine different HLA-A2.1- and A11-restricted epitopes derived from HBVand HIV induced CTL against all epitopes (Ishioka et al., J Immunol,Vol. 162(7):3915-25 (1999)).

Thus, minigene vaccines containing multiple MHC Class I and Class II(i.e., CTL and HTL) epitopes can be designed, and presentation andrecognition can be obtained for all epitopes. However, theimmunogenicity of multi-epitope constructs appears to be stronglyinfluenced by a number of variables, a number of which have heretoforebeen unknown. For example, the immunogenicity (or antigenicity) of thesame epitope expressed in the context of different vaccine constructscan vary over several orders of magnitude. Thus, there exists a need toidentify strategies to optimize multi-epitope vaccine constructs. Suchoptimization is important in terms of induction of potent immuneresponses and ultimately, for clinical efficacy. Accordingly, thepresent invention provides strategies to optimize antigenicity andimmunogenicity of multi-epitope vaccines encompassing a large number ofepitopes, and optimized multi-epitope vaccines, particularly minigenevaccines, generated in accordance with these strategies.

The following paragraphs provide a brief review of some of the mainvariables potentially influencing minigene immunogenicity, epitopeprocessing, and presentation on antigen presenting cells (APCs) inassociation with Class I and Class II MHC molecules.

Immunodominance

Of the many thousand possible peptides that are encoded by a complexforeign pathogen, only a small fraction ends up in a peptide formcapable of binding to MHC Class I antigens and thus of being recognizedby T cells. This phenomenon, of obvious potential impact on thedevelopment of a multi-epitope vaccine, is known as immunodominance(Yewdell et al., Annu Rev Immunol, 17:51-88 (1999)). Several majorvariables contribute to immunodominance. Herein, we describe variablesaffecting the generation of the appropriate peptides, both inqualitative and quantitative terms, as a result of intracellularprocessing.

Junctional Epitopes

A junctional epitope is defined as an epitope created due to thejuxtaposition of two other epitopes. The new epitope is composed of aC-terminal section derived from a first epitope, and an N-terminalsection derived from a second epitope. Creation of junctional epitopesis a potential problem in the design of multi-epitope minigene vaccines,for both Class I and Class II restricted epitopes for the followingreasons. Firstly, when developing a minigene composed of, or containing,human epitopes, which are typically tested for immunogenicity in HLAtransgenic laboratory animals, the creation of murine epitopes couldcreate undesired immunodominance effects. Secondly, the creation of new,unintended epitopes for human HLA Class I or Class II molecules couldelicit in vaccine recipients, new T cell specificities that are notexpressed by infected cells or tumors that are targets of induced T cellresponses. These responses are by definition irrelevant and ineffectiveand could even be counterproductive, by creating undesiredimmunodominance effects.

The existence of junctional epitopes has been documented in a variety ofdifferent experimental situations. Gefter and collaborators firstdemonstrated the effect in a system in which two different Class IIrestricted epitopes were juxtaposed and colinearly synthesized (Perkinset al., J Immunol, Vol. 146(7):2137-44 (1991)). The effect was so markedthat the immune system recognition of the epitopes could be completely“silenced” by these new junctional epitopes (Wang et al., Cell Immunol,Vol. 143(2):284-97 (1992)). Helper T cells directed against junctionalepitopes were also observed in humans as a result of immunization with asynthetic lipopeptide, which was composed of an HLA-A2-restrictedHBV-derived immunodominant CTL epitope, and a universal TetanusToxoid-derived HTL epitope (Livingston et al, J Immunol, Vol.159(3):1383-92 (1997)). Thus, the creation of junctional epitopes is amajor consideration in the design of multi-epitope constructs.

The present invention provides methods of addressing this problem andavoiding or minimizing the occurrence of junctional epitopes.

Flanking Regions

Class I restricted epitopes are generated by a complex process (Yewdellet al., Annu Rev Immunol, 17:51-88 (1999)). Limited proteolysisinvolving endoproteases and potential trimming by exoproteases isfollowed by translocation across the endoplasmic reticulum (ER) membraneby transporters associated with antigen processing (TAP) molecules. Themajor cytosolic protease complex involved in generation of antigenicpeptides, and their precursors, is the proteasome (Niedermann et al.,Immunity, Vol. 2(3):289-99 (1995)), although ER trimming of CTLprecursors has also been demonstrated (Paz et al., Immunity Vol.11(2):241-51 (1999)). It has long been debated whether or not theresidues immediately flanking the C and N terminus of the epitope, havean influence on the efficiency of epitope generation.

The yield and availability of processed epitope has been implicated as amajor variable in determining immunogenicity and could thus clearly havea major impact on overall minigene potency in that the magnitude ofimmune response can be directly proportional to the amount of epitopebound by MHC and displayed for T cell recognition. Several studies haveprovided evidence that this is indeed the case. For example, inductionof virus-specific CTL that is essentially proportional to epitopedensity (Wherry et al., J Immunol, Vol. 163(7):3735-45 (1999)) has beenobserved. Further, recombinant minigenes, which encode a preprocessedoptimal epitope, have been used to induce higher levels of epitopeexpression than naturally observed with full-length protein (Anton etal., J Immunol, Vol. 158(6):2535-42 (1997)). In general, minigenepriming has been shown to be more effective than priming with the wholeantigen (Restifo et al., J Immunol, Vol. 154(9):4414-22 (1995); Ishiokaet al., J Immunol, Vol. 162(7):3915-25 (1999)), even though someexceptions have been noted (Iwasaki et al., Vaccine, Vol.17(15-16):2081-8 (1999)).

Early studies concluded that residues within the epitope (Hahn et al., JExp Med, Vol. 176(5):1335-41 (1992)) primarily regulate immunogenicity.Similar conclusions were reached by other studies, mostly based ongrafting an epitope in an unrelated gene, or in the same gene, but in adifferent location (Chimini et al., J Exp Med, Vol. 169(1):297-302(1989); Hahn et al., J Exp Med, Vol. 174(3):733-6 (1991)). Otherexperiments however (Del Val et al., Cell, Vol. 66(6):1145-53 (1991);Hahn et al., J Exp Med, Vol. 176(5):1335-41 (1992)), suggested thatresidues localized directly adjacent to the CTL epitope can directlyinfluence recognition (Couillin et al., J Exp Med, Vol. 180(3):1129-34(1994); Bergmann et al., J. Virol. Vol. 68(8):5306-10 (1994)). In thecontext of minigene vaccines, the controversy has been renewed. Shastriand coworkers (Shastri et al., J Immunol, Vol. 155(9):4339-46 (1995))found that T cell responses were not significantly affected by varyingthe N-terminal flanking residue but were inhibited by the addition of asingle C-terminal flanking residue. The most dramatic inhibition wasobserved with isoleucine, leucine, cysteine, and proline as theC-terminal flanking residues. In contrast, Gileadi (Gileadi et al., EurJ Immunol, Vol. 29(7):2213-22 (1999)) reported profound effects as afunction of the residues located at the N terminus of mouse influenzavirus epitopes. Bergmann and coworkers found that aromatic, basic andalanine residues supported efficient epitope recognition, while G and Presidues were strongly inhibitory (Bergmann et al., J Immunol, Vol.157(8):3242-9 (1996)). In contrast, Lippolis (Lippolis et al., J Virol,Vol. 69(5):3134-46 (1995)) concluded that substituting flanking residuesdid not effect recognition. However, only rather conservativesubstitutions that are unlikely to affect proteasome specificity weretested.

It appears that the specificity of these effects, and in general ofnatural epitopes, roughly correlates with proteasome specificity. Forexample, proteasome specificity is partly trypsin-like (Niedermann etal., Immunity, Vol. 2(3):289-99 (1995)), with cleavage following basicamino acids. Nevertheless, efficient cleavage of the carboxyl side ofhydrophobic and acidic residues is also possible. Consistent with thesespecificities are the studies of Sherman and collaborators, which foundthat an R to H mutation at the position following the C-terminus of ap53 epitope affects proteasome-mediated processing of the protein(Theobald et al., J Exp Med, Vol. 188(6):1017-28 (1998)). Several otherstudies (Hanke et al., J Gen Virol, Vol. 79 (Pt 1):83-90 (1998); Thomsonet al., Proc Natl Acad Sci USA, Vol. 92(13):5845-9 (1995)) indicatedthat minigenes can be constructed utilizing minimal epitopes, and thatthese flanking sequences appear not be required, although the potentialfor further optimization by the use of flanking regions was alsoacknowledged.

In sum, for HLA Class I epitopes, the effects of flanking regions onprocessing and presentation of CTL epitopes is as yet undefined. Asystematic analysis of the effect of modulation of flanking regions hasnot been performed for minigene vaccines. Thus, analysis utilizingminigene vaccines encoding epitopes restricted by human Class I ingeneral is needed. The present invention provides such an analysis andaccordingly, provides multi-epitope vaccine constructs optimized forimmunogenicity and antigenicity, and methods of designing suchconstructs.

HLA Class II peptide complexes are also generated as a result of acomplex series of events that is distinct from HLA Class I processing.The processing pathway involves association with Invariant chain (Ii),its transport to specialized compartments, the degradation of Ii toCLIP, and HLA-DM catalyzed removal of CLIP (see (Blum et al., Crit RevImmunol, Vol. 17(5-6):411-7 (1997); Arndt et al., Immunol Res, Vol.16(3):261-72 (1997)) for review. Moreover, there is a potentiallycrucial role of various cathepsins in general, and cathepsin S and L inparticular, in Ii degradation (Nakagawa et al., Immunity, Vol.10(2):207-17 (1999)). In terms of generation of functional epitopeshowever, the process appears to be somewhat less selective (Chapman H.A., Curr Opin Immunol, Vol. 10(1):93-102 (1998)), and peptides of manysizes can bind to MHC Class II (Hunt et al., Science, Vol.256(5065):1817-20 (1992)). Most or all of the possible peptides appearto be generated (Moudgil et al., J Immunol, Vol. 159(6):2574-9 (1997);and Thomson et al., J Virol, Vol. 72(3):2246-52 (1998)). Thus, ascompared to the issue of flanking regions, the creation of junctionalepitopes can be a more serious concern in particular embodiments.

SUMMARY OF THE INVENTION

The invention provides multi-epitope nucleic acid constructs encoding aplurality of CTL and/or HTL epitopes and polypeptide constructscomprising a plurality of CTL and/or HTL epitopes (preferably encoded bythe nucleic acid constructs), as well as cells comprising such nucleicacid constructs and/or polypeptide constructs, compositions comprisingsuch nucleic acid constructs and/or polypeptide constructs and/or suchcells, and methods for stimulating an immune response (e.g. therapeuticmethods) utilizing such nucleic acid constructs and/or polypeptideconstructs and/or compositions and/or cells.

In some embodiments, the invention provides a polynucleotide comprisingor alternatively consisting of:

-   -   (a) a multi-epitope construct (e.g., minigene) comprising        nucleic acids encoding the hepatitis B virus (HBV) cytotoxic T        lymphocyte (CTL) epitopes pol 562, pol 745, env 332, pol 530,        pol 388, env 249, env 359, pol 640, env 335, env 183, env 313,        core 117, core 19, core 18, core 419, pol 392, pol 531, pol 415,        pol 47, pol 455, core 141, pol 429, env 236, pol 166, pol 538,        core 101, pol 354 and core 137 (i.e., the HBV CTL epitope each        consisting of the relevant sequence in Table 7), wherein the        nucleic acids are directly or indirectly joined to one another        in the same reading frame;    -   (b) the multi-epitope construct of (a), which further comprises        a nucleic acid encoding the HBV CTL epitope pol 665 (i.e. the        pol 665 epitope in Table 7), directly or indirectly joined in        the same reading frame to CTL epitope nucleic acids of (a);    -   (c) a multi-epitope construct comprising nucleic acids encoding        the hepatitus B virus (HBV) cytotoxic T lymphocyte (CTL)        epitopes pol 149, core 18, pol 562, pol 538, pol 455, env 183,        core 141, pol 665, env 335, env 313, pol 354, pol 629, core 19,        pol 150, pol 47, pol 388, pol 531 and pol 642, wherein the        nucleic acids are directly or indirectly joined to one another        in the same reading frame;    -   (d) the multi-epitope construct of (a) or (b) or (c), which        further comprises one or a plurality of spacer nucleic acids,        directly or indirectly joined in the same reading frame to the        CTL epitope nucleic acids;    -   (e) the multi-epitope construct of (d), wherein the one or        plurality of spacer nucleic acids are positioned between the CTL        epitope nucleic acids of (a), between the CTL epitope nucleic        acids of (a) and (b), between the CTL epitope nucleic acids        of (a) and (b) and of (a) and of (c), or between the CTL epitope        nucleic acids of (c);    -   (f) the multi-epitope construct of (d) or (e), wherein the        spacer nucleic acids encode an amino acid sequence 1 to 8        residues in length;    -   (g) the multi-epitope construct of any of (d) to (f), wherein        two or more of the spacer nucleic acids encode different (i.e.,        non-identical) amino acid sequences;    -   (h) the multi-epitope construct of any of (d) to (g), wherein        two or more of the spacer nucleic acids encode an amino acid        sequence different from the amino acid sequence encoded by other        spacer nucleic acids;    -   (i) the multi-epitope construct of any of (d) to (h), wherein        two or more of the spacer nucleic acids encodes the identical        amino acid sequence;    -   (j) the multi-epitope construct of any of (d) to (i), wherein        one or more of the spacer nucleic acids encode an amino acid        sequence comprising or consisting of three consecutive alanine        (Ala) residues;    -   (k) the multi-epitope construct of any of (a) to (j), which        further comprises one or a plurality of nucleic acids encoding a        HTL epitope, directly or indirectly joined in the same reading        frame to the CTL epitope nucleic acids and/or the spacer nucleic        acids;    -   (l) the multi-epitope construct of (k), wherein the HTL epitope        is a PADRE® epitope;    -   (m) the multi-epitope construct of (k), wherein the HTL epitope        is an HBV HTL epitope;    -   (n) the multi-epitope construct of (m), wherein the HBV HTL        epitope is selected from the group consisting of pol 774, pol        694, pol 145, core 50, pol 385, pol 523, env 339, pol 501, pol        420, pol 412, env 180, core 120, pol 96, pol 618, pol 767, and        pol 664 (i.e., the HBV HTL epitope each consisting of the        relevant sequence in Table 11);    -   (o) the multi-epitope construct of any of (k) to (n), which        further comprises one or a plurality of spacer nucleic acids        between a CTL epitope and an HTL epitope or between HTL        epitopes;    -   (p) the multi-epitope construct of any of (a) to (O), which        further comprises one or more MHC Class I and/or MHC Class II        targeting nucleic acid;    -   (q) the multi-epitope construct of (p), wherein the targeting        nucleic acid encodes a targeting sequence selected from the        group consisting of: Ig kappa signal sequence, tissue        plasminogen activator signal sequence, insulin signal sequence,        endoplasmic reticulum signal sequence, LAMP-1 lysosomal        targeting sequence, LAMP-2 lysosomal targeting sequence, HLA-DM        lysosomal targeting sequence, HLA-DM-association sequences of        HLA-DO, Ig-α cytoplasmic domain, Ig-β cytoplasmic domain, Ii        protein, influenza matrix protein, HBV surface antigen, HBV core        antigen, and yeast Ty protein;    -   (r) the multi-epitope construct of any of (a) to (q), which is        optimized for CTL and/or HTL epitope processing;    -   (s) the multi-epitope construct of any of (a) to (r), wherein        the CTL nucleic acids are sorted to minimize the number of CTL        and/or HTL junctional epitopes;    -   (t) the multi-epitope construct of any of (k) to (s), wherein        the HTL nucleic acids are sorted to minimize the number of CTL        and/or HTL junctional epitopes;    -   (u) the multi-epitope construct of any of (a) to (t), which        comprises one or more nucleic acids encoding one or more        flanking amino acid residues;    -   (v) the multi-epitope construct of (u), wherein the one or more        flanking amino acid residues is selected from the group        consisting of: K, R, N, Q, G, A, S, C, and T at a C+1 position        of a CTL epitope nucleic acid;    -   (w) the multi-epitope construct of any of (a) to (v), wherein        the HBV CTL nucleic acids are joined in the order shown in FIG.        27A;    -   (x) the multi-epitope construct of any of (n) to (w), wherein        the HBV HTL nucleic acids are joined in the order shown in FIG.        28A;    -   (y) the multi-epitope construct of any of (c) to (v) or (x),        wherein the HBV CTL nucleic acids are joined in the order shown        in FIG. 34.    -   (z) the multi-epitope construct of any of (a) to (x), which        encodes a peptide comprising or consisting of an amino acid        sequence shown in FIG. 24B, or Table 13, 14, 18 or 19;    -   (aa) the multi-epitope construct of (z), which comprises a        nucleic acid sequence selected from the group consisting of:        nucleotides +1 to 1248 of the nucleotide sequence in Table 13,        nucleotides +1 to 1032 of the nucleotide sequence in Table 14,        the nucleotide sequence in FIG. 24C, nucleotides +1 to 2292 of        the nucleotide sequence in Table 18, and nucleotides +1 to 2232        of the nucleotide sequence in Table 19;    -   (bb) the multi-epitope construct of any of (c) to (v) or (x)        or (y) or (z), which encodes a peptide comprising or consisting        of an amino acid sequence shown in Table 23 or 24;    -   (cc) the multi-epitope construct of (bb), which comprises a        nucleic acid sequence selected from the group consisting of:        nucleotides +1 to 618 of the nucleotide sequence in Table 23, or        nucleotides +1 to 657 of the nucleotide sequence in Table 24;    -   (dd) the multi-epitope construct of any of (a) to (cc), and one        or more regulatory sequences;    -   (ee) the multi-epitope construct of any of (a) to (dd), and one        or more IRESs;    -   (ff) the multi-epitope construct of any of (a) to (ee), and one        or more promoters;    -   (gg) the multi-epitope construct of any of (a) to (ff), and one        or more CMV promoters;    -   (hh) the multi-epitope construct of any of (a) to (gg), and two        or more CMV promoters;    -   (ii) the multi-epitope construct of any of (a) to (hh), and a        vector;    -   (jj) the multi-epitope construct of (ii), wherein the vector is        an expression vector;    -   (kk) the multi-epitope construct of any of (a) to (jj), which        has the structure of a multi-epitope construct shown in FIG.        29A(i), (ii), or (iii).

In some embodiments, the polynucleotide of (a) to (kk) has the structureof a vector shown in FIG. 29A(i), (ii), or (iii).

In some embodiments, the invention provides a polynucleotide comprisingtwo multi-epitope constructs, the first comprising the HBV multi-epitopeconstruct in any of (a) to (kk), above, and the second comprising HBVHTL epitopes such as those in (n), wherein the first and secondmulti-epitope constructs are not directly joined, and/or are not joinedin the same frame. Each first and second multi-epitope construct may beoperably linked to a regulatory sequence such as a promoter or an IRES.The polynucleotide comprising the first and second multi-epitopecontructs may comprise, e.g., at least one promoter and at least oneIRES, one promoter and one IRES, two promoters, or two or more promotersand/or IRESs. The promoter may be a CMV promoter or other promoterdescribed herein or known in the art. In preferred embodiments, the twomulti-epitope constructs have the structure shown in FIG. 29A(i) or(ii). The second multi-epitope construct may encode a peptide comprisingor consisting of an amino acid sequence shown in FIG. 24C or Table 14.The second multi-epitope construct may comprises a nucleic acid sequenceselected from the nucleotide sequence in FIG. 24C, and nucleotides +1 to1032 of the nucleotide sequence in Table 14.

In other embodiments the invention provides peptides encoded by thepolynucleotides described above, for example, a peptide comprising oralternatively consisting of:

-   -   (a) a multi-epitope construct (e.g., minigene) comprising the        hepatitis B virus (HBV) cytotoxic T lymphocyte (CTL) epitopes        pol 562, pol 745, env 332, pol 530, pol 388, env 249, env 359,        pol 640, env 335, env 183, env 313, core 117, core 19, core 18,        core 419, pol 392, pol 531, pol 415, pol 47, pol 455, core 141,        pol 429, env 236, pol 166, pol 538, core 101, pol 354 and core        137 (i.e., CTL epitopes of FIG. 27A, consisting of the sequences        in Table 7), directly or indirectly joined to one another;    -   (b) the multi-epitope construct of (a), which further comprises        the HBV CTL epitope pol 665, directly or indirectly joined to        the CTL epitopes of (a);    -   (c) a multi-epitope construct comprising the hepatitus B virus        (HBV) cytotoxic T lymphocyte (CTL) epitopes pol 149, core 18,        pol 562, pol 538, pol 455, env 183, core 141, pol 665, env 335,        env 313, pol 354, pol 629, core 19, pol 150, pol 47, pol 388,        pol 531 and pol 642, directly or indirectly joined to one        another;    -   (d) the multi-epitope construct of (a) or (b) or (c), which        further comprises one or a plurality of spacers, directly or        indirectly joined to the CTL epitopes;    -   (e) the multi-epitope construct of (d), wherein the one or        plurality of spacers are positioned between the CTL epitopes of        (a), between the CTL epitopes of (a) and (b), between the CTL        epitopes of (a) and (b) and of (a) and of (c), or between the        CTL epitopes of (c);    -   (f) the multi-epitope construct of (d) or (e), wherein the        spacers are 1 to 8 amino acid residues in length;    -   (g) the multi-epitope construct of any of (d) to (f), wherein        two or more of the spacers comprise or consist of different        (i.e., non-identical) amino acid sequences;    -   (h) the multi-epitope construct of any of (d) to (g), wherein        two or more of the spacers comprise or consist of an amino acid        sequence different from the amino acid sequence of the other        spacers;    -   (i) the multi-epitope construct of any of (d) to (h), wherein        two or more of the spacers comprise or consist of the identical        amino acid sequence;    -   (j) the multi-epitope construct of any of (d) to (i), wherein        one or more of the spacers comprises or consists of three        consecutive alanine (Ala) residues;    -   (k) the multi-epitope construct of any of (a) to (O), which        further comprises one or a plurality of HTL epitopes, directly        or indirectly joined to the CTL epitopes and/or the spacers;    -   (l) the multi-epitope construct of (k), wherein the one or        plurality of HTL epitopes is a PADRE® epitope;    -   (m) the multi-epitope construct of (k), wherein the one or        plurality of HTL epitopes is an HBV HTL epitope;    -   (n) the multi-epitope construct of (m), wherein the one or        plurality of HTL epitopes is selected from the group consisting        of pol 774, pol 694, pol 145, core 50, pol 385, env 339, pol        501, pol 420, pol 412, env 180, core 120, pol 96, pol 618, pol        767, and pol 664;    -   (o) the multi-epitope construct of any of (k) to (n), which        further comprises one or a plurality of spacers between a CTL        epitope and an HTL epitope or between HTL epitopes;    -   (p) the multi-epitope construct of any of (a) to (O), which        further comprises one or more MHC Class I and/or MHC Class II        targeting sequences;    -   (q) the multi-epitope construct of (p), wherein the one or more        targeting sequence is selected from the group consisting of: Ig        kappa signal sequence, tissue plasminogen activator signal        sequence, insulin signal sequence, and endoplasmic reticulum        signal sequence, LAMP-1 lysosomal targeting sequence, LAMP-2        lysosomal targeting sequence, HLA-DM lysosomal targeting        sequence, HLA-DM-association sequences of HLA-DO, Ig-α        cytoplasmic domain, Ig-β cytoplasmic domain, Ii protein,        influenza matrix protein, HBV surface antigen, HBV core antigen,        and yeast Ty protein;    -   (r) the multi-epitope construct of any of (a) to (q), which is        optimized for CTL and/or HTL epitope processing;    -   (s) the multi-epitope construct of any of (a) to (r), wherein        the CTL epitopes are sorted to minimize the number of CTL and/or        HTL junctional epitopes;    -   (t) the multi-epitope construct of any of (k) to (s), wherein        the HTL epitopes are sorted to minimize the number of CTL and/or        HTL junctional epitopes;    -   (u) the multi-epitope construct of any of (a) to (t), which        comprises one or more flanking amino acid residues;    -   (v) the multi-epitope construct of (u), wherein one or more the        flanking amino acid residues is selected from the group        consisting of: K, R, N, Q, G, A, S, C, and T at a C+1 position        of a CTL epitope;    -   (w) the multi-epitope construct of any of (a) to (v), wherein        the HBV CTL epitopes are joined in the order shown in FIG. 27A;    -   (x) the multi-epitope construct of any of (n) to (w), wherein        the HBV HTL epitopes are joined in the order shown in FIG. 28A;    -   (y) the multi-epitope construct of any of (c) to (v) or (x),        wherein the HBV CTL epitopes are joined in the order shown in        FIG. 34;    -   (z) the multi-epitope construct of any of (a) to (x), which        comprises or consists of an amino acid sequence shown in FIG.        24B, or Table 13, 14, 18 or 19;    -   (aa) the multi-epitope construct of (z), which is encoded by a        nucleic acid sequence selected from the group consisting of:        nucleotides +1 to 1248 of the nucleotide sequence in Table 13,        nucleotides +1 to 1032 of the nucleotide sequence in Table 14,        the nucleotide sequence in FIG. 24C, nucleotides +1 to 2292 of        the nucleotide sequence in Table 18, and nucleotides +1 to 2232        of the nucleotide sequence in Table 19;    -   (bb) the multi-epitope construct of any of (c) to (v), or (x) or        (y), which comprises or consists of an amino acid sequence shown        in Table 23 or 24;    -   (cc) the multi-epitope construct of (bb), which is encoded by a        nucleic acid sequence selected from the group consisting of:        nucleotides +1 to 618 of the nucleotide sequence in Table 23 and        nucleotides +1 to 657 of the nucleotide sequence in Table 24.

In other embodiments, the invention provides cells comprising thepolynucleotides and/or polypeptides above; compositions comprising thepolynucleotides and/or polypeptides and/or cells; methods for makingthese polynucleotides, polypeptides, cells and compositions; and methodsfor stimulating an immune response (e.g. therapeutic and/or prophylacticmethods) utilizing these polynucleotides and/or polypeptides and/orcells and/or compositions. The invention is described in further detailbelow.

Definitions

The following definitions are provided to enable one of ordinary skillin the art to understand some of the preferred embodiments of inventiondisclosed herein. It is understood, however, that these definitions areexemplary only and should not be used to limit the scope of theinvention as set forth in the claims. Those of ordinary skill in the artwill be able to construct slight modifications to the definitions belowand utilize such modified definitions to understand and practice theinvention disclosed herein. Such modifications, which would be obviousto one of ordinary skill in the art, as they may be applicable to theclaims set forth below, are considered to be within the scope of thepresent invention.

Throughout this disclosure, “binding data” results are often expressedin terms of “IC₅₀'s.” IC₅₀ is the concentration of peptide in a bindingassay at which 50% inhibition of binding of a reference peptide isobserved. Given the conditions in which the assays are run (i.e.,limiting HLA proteins and labeled peptide concentrations), these valuesapproximate K_(D) values. Assays for determining binding are describedin detail, e.g., in PCT publications WO 94/20127 and WO 94/03205. Itshould be noted that IC₅₀ values can change, often dramatically, if theassay conditions are varied, and depending on the particular reagentsused (e.g., HLA preparation, etc.). For example, excessiveconcentrations of HLA molecules will increase the apparent measured IC₅₀of a given ligand. Alternatively, binding is expressed relative to areference peptide. Although as a particular assay becomes more, or less,sensitive, the IC₅₀'s of the peptides tested may change somewhat, thebinding relative to the reference peptide will not significantly change.For example, in an assay run under conditions such that the IC₅₀ of thereference peptide increases 10-fold, the IC₅₀ values of the testpeptides will also shift approximately 10-fold. Therefore, to avoidambiguities, the assessment of whether a peptide is a good,intermediate, weak, or negative binder is generally based on its IC₅₀,relative to the IC₅₀ of a standard peptide. Binding may also bedetermined using other assay systems including those using: live cells(e.g., Ceppellini et al., Nature 339:392, 1989; Christnick et al.,Nature 352:67, 1991; Busch et al., Int. Immunol. 2:443, 19990; Hill etal., J. Immunol. 147:189, 1991; del Guercio et al., J. Immunol. 154:685,1995), cell free systems using detergent lysates (e.g., Cerundolo etal., J. Immunol. 21:2069, 1991), immobilized purified MHC (e.g., Hill etal., J. Immunol. 152, 2890, 1994; Marshall et al., J. Immunol. 152:4946,1994), ELISA systems (e.g., Reay et al., EMBO J. 11:2829, 1992), surfaceplasmon resonance (e.g., Khilko et al., J. Biol. Chem. 268:15425, 1993);high flux soluble phase assays (Hammer et al., J. Exp. Med. 180:2353,1994), and measurement of class I MHC stabilization or assembly (e.g.,Ljunggren et al., Nature 346:476, 1990; Schumacher et al., Cell 62:563,1990; Townsend et al., Cell 62:285, 1990; Parker et al., J. Immunol.149:1896, 1992).

The designation of a residue position in an epitope as the “carboxylterminus” or the “carboxyl terminal position” refers to the residueposition at the end of the epitope that is nearest to the carboxylterminus of a peptide, which is designated using conventionalnomenclature as defined below. “C+1” refers to the residue or positionimmediately following the C-terminal residue of the epitope, i.e.,refers to the residue flanking the C-terminus of the epitope. The“carboxyl terminal position” of the epitope occurring at the carboxylend of the multi-epitope construct may or may not actually correspond tothe carboxyl terminal end of polypeptide. In preferred embodiments, theepitopes employed in the optimized multi-epitope constructs aremotif-bearing epitopes and the carboxyl terminus of the epitope isdefined with respect to primary anchor residues corresponding to aparticular motif.

The designation of a residue position in an epitope as “amino terminus”or “amino-terminal position” refers to the residue position at the endof the epitope which is nearest to the amino terminus of a peptide,which is designated using conventional nomenclature as defined below.“N−1” refers to the residue or position immediately adjacent to theepitope at the amino terminal end (position number 1) of an eptiope. The“amino terminal position” of the epitope occurring at the amino terminalend of the multi-epitope construct may or may not actually correspondsto the amino terminal end of the polypeptide. In preferred embodiments,the epitopes employed in the optimized multi-epitope constructs aremotif-bearing epitopes and the amino terminus of the epitope is definedwith respect to primary anchor residues corresponding to a particularmotif.

A “computer” or “computer system” generally includes: a processor; atleast one information storage/retrieval apparatus such as, for example,a hard drive, a disk drive or a tape drive; at least one input apparatussuch as, for example, a keyboard, a mouse, a touch screen, or amicrophone; and display structure. Additionally, the computer mayinclude a communication channel in communication with a network suchthat remote users may communicate with the computer via the network toperform multi-epitope construct optimization functions disclosed herein.Such a computer may include more or less than what is listed above. Thenetwork may be a local area network (LAN), wide area network (WAN) or aglobal network such as the world wide web (e.g., the internet).

A “construct” as used herein generally denotes a composition that doesnot occur in nature. A construct may be a “polynucleotide construct” ora “polypeptide construct.” A construct can be produced by synthetictechnologies, e.g., recombinant DNA preparation and expression orchemical synthetic techniques for nucleic acids and amino acids andpepetides and polypeptides. A construct can also be produced by theaddition or affiliation of one material with another such that theresult is not found in nature in that form.

The term “multi-epitope construct” when referring to nucleic acids andpolynucleotides can be used interchangeably with the terms “minigene”and “multi-epitope nucleic acid vaccine,” and other equivalent phrases,and comprises multiple epitope nucleic acids that encode peptideepitopes of any length that can bind to a molecule functioning in theimmune system, preferably a class I HLA and a T-cell receptor or a classII HLA and a T-cell receptor. The epitope nucleic acids in amulti-epitope construct can encode class I HLA epitopes and/or class IIHLA epitopes. Class I HLA-encoding epitope nucleic acids are referred toas CTL epitope nucleic acids, and class II HLA-encoding epitope nucleicacids are referred to as HTL epitope nucleic acids. Some multi-epitopeconstructs can have a subset of the multi-epitope nucleic acids encodingclass I HLA epitopes and another subset of the multi-epitope nucleicacids encoding class II HLA epitopes. The CTL epitope nucleic acidspreferably encode an epitope peptide of less than about 15 residues inlength, or less than about 13 amino acids in length, or less than about11 amino acids in length, preferably about 8 to about 13 amino acids inlength, more preferably about 8 to about 11 amino acids in length (e.g.8, 9, 10, or 11), and most preferably about 9 or 10 amino acids inlength. The HTL epitope nucleic acids can encode an epitope peptide ofless than about 50 residues in length, and usually consist of about 6 toabout 30 residues, more usually between about 12 to 25, and often about15 to 20, and preferably about 7 to about 23, preferably about 7 toabout 17, more preferably about 11 to about 15 (e.g. 11, 12, 13, 14, or15), and most preferably about 13 amino acids in length. Themulti-epitope constructs described herein preferably include 5 or more,10 or more, 15 or more, 20 or more, or 25 or more epitope nucleic acids.All of the epitope nucleic acids in a multi-epitope construct may befrom one organism (e.g., the nucleotide sequence of every epitopenucleic acid may be present in HBV or HIV strains), or the multi-epitopeconstruct may include epitope nucleic acids sequences present in two ormore different organisms (e.g., the nucleotide sequence of_some epitopeencoding nucleic acid sequences from HBV and some from HIV and/or somefrom HCV). The term “epigene” is used herein to refer to certainmulti-epitope constructs. As described hereafter, one or more epitopenucleic acids in the multi-epitope construct may be flanked by a spacernucleic acid, and/or other nucleic acids also described herein orotherwise known in the art.

The term “multi-epitope construct,” when referring to polypeptides, canbe used interchangeably with the terms “minigene construct,”“multi-epitope vaccine,” and other equivalent phrases, and comprisesmultiple peptide epitopes of any length that can bind to a moleculefunctioning in the immune system, preferably a class I HLA and a T-cellreceptor or a class II HLA and a T-cell receptor. The epitopes in amulti-epitope construct can be class I HLA epitopes and/or class II HLAepitopes. Class I HLA epitopes are referred to as CTL epitopes, andclass II HLA epitopes are referred to as HTL epitopes. Somemulti-epitope constructs can have a subset of class I HLA epitopes andanother subset of class II HLA epitopes. The CTL epitopes preferably areless than about 15 residues in length, or less than about 13 residues inlength, or less than about 11 residues in length, and preferably encodean epitope peptide of about 8 to about 13 amino acids in length, morepreferably about 8 to about 11 amino acids in length (e.g. 8, 9, 10, or11), and most preferably about 9 amino acids in length. The HTL epitopesare less than about 50 residues in length and usually consist of about 6to about 30 residues, more usually between about 12 to 25, and oftenabout 15 to 20 residues, and preferably about 7 to about 23, preferablyabout 7 to about 17, more preferably about 11 to about 15 (e.g. 11, 12,13, 14, or 15), and most preferably about 13 amino acids in length. Themulti-epitope constructs described herein preferably include 5 or more,10 or more, 15 or more, 20 or more, or 25 or more epitopes. All of theepitopes in a multi-epitope construct may be from one organism (e.g.,every epitope may be present in HBV or HIV strains), or themulti-epitope construct may include epitopes present in two or moredifferent organisms (e.g., some epitopes from HBV and some from HIVand/or some from HCV). The term “epigene” is used herein to refer tocertain multi-epitope constructs. As described hereafter, one or moreepitopes in the multi-epitope construct may be flanked by a spacersequences, and/or other sequences also described herein or otherwiseknown in the art.

A “multi-epitope vaccine,” which is synonyous with a “polyepitopicvaccine,” is a vaccine comprising multiple epitopes.

“Cross-reactive binding” indicates that a peptide is bound by more thanone HLA molecule; a synonym is “degenerate binding.”

A “cryptic epitope” elicits a response by immunization with an isolatedpeptide, but the response is not cross-reactive in vitro when intactwhole protein that comprises the epitope is used as an antigen.

A “dominant epitope” is an epitope that induces an immune response uponimmunization with a whole native antigen (see, e.g., Sercarz, et al.,Annu. Rev. Immunol. 11:729-766, 1993). Such a response is cross-reactivein vitro with an isolated peptide epitope.

With regard to a particular amino acid sequence, an “epitope” is a setof amino acid residues that is involved in recognition by a particularimmunoglobulin, or in the context of T cells, those residues necessaryfor recognition by T cell receptor proteins and/or MajorHistocompatibility Complex (MHC) receptors. In an immune system setting,in vitro or in vivo, an epitope is the collective features of amolecule, such as primary, secondary and tertiary peptide structure, andcharge, that together form a site recognized by an immunoglobulin, Tcell receptor or HLA molecule. Throughout this disclosure epitope andpeptide are often used interchangeably. It is to be appreciated,however, that isolated or purified protein or peptide molecules largerthan and comprising an epitope of the invention are still within thebounds of the invention.

A “flanking residue” is a residue that is positioned next to an epitope.A flanking residue can be introduced or inserted at a position adjacentto the N-terminus or the C-terminus of an epitope.

An “immunogenic peptide” or “peptide epitope” is a peptide thatcomprises an allele-specific motif or supermotif such that the peptidewill bind an HLA molecule and induce a CTL and/or HTL response. Thus,immunogenic peptides of the invention are capable of binding to anappropriate HLA molecule and thereafter inducing a cytotoxic T cellresponse, or a helper T cell response, to the antigen from which theimmunogenic peptide is derived.

“Heteroclitic analogs” are defined herein as a peptide with increasedpotency for a specific T cell, as measured by increased responses to agiven dose, or by a requirement of lesser amounts to achieve the sameresponse. Advantages of heteroclitic analogs include that the epitopescan be more potent, or more economical (since a lower amount is requiredto achieve the same effect). In addition, modified epitopes mightovercome antigen-specific T cell unresponsiveness (T cell tolerance).

“Human Leukocyte Antigen” or “HLA” is a human class I or class II MajorHistocompatibility Complex (MHC) protein (see, e.g., Stites, et al.,IMMUNOLOGY, 8^(TH) ED., Lange Publishing, Los Altos, Calif. (1994)).

An “HLA supertype or HLA family,” as used herein, describes sets of HLAmolecules grouped based on shared peptide-binding specificities. HLAclass I molecules that share similar binding affinity for peptidesbearing certain amino acid motifs are grouped into such HLA supertypes.The terms HLA superfamily, HLA supertype family, HLA family, and HLAxx-like molecules (where xx denotes a particular HLA type), aresynonyms.

As used herein, “high affinity” with respect to HLA class I molecules isdefined as binding with an IC₅₀, or K_(D) value, of 50 nM or less;“intermediate affinity” with respect to HLA class I molecules is definedas binding with an IC₅₀ or K_(D) value of between about 50 and about 500nM. “High affinity” with respect to binding to HLA class II molecules isdefined as binding with an IC₅₀ or K_(D) value of 100 nM or less;“intermediate affinity” with respect to binding to HLA class IImolecules is defined as binding with an IC₅₀ or K_(D) value of betweenabout 100 and about 1000 nM.

An “IC₅₀” is the concentration of peptide in a binding assay at which50% inhibition of binding of a reference peptide is observed. Dependingon the conditions in which the assays are run (i.e., limiting HLAproteins and labeled peptide concentrations), these values mayapproximate K_(D) values.

The terms “identical” or percent “identity,” in the context of two ormore peptide sequences, refer to two or more sequences or subsequencesthat are the same or have a specified percentage of amino acid residuesthat are the same, when compared and aligned for maximum correspondenceover a comparison window, as measured using a sequence comparisonalgorithm or by manual alignment and visual inspection.

“Introducing” an amino acid residue at a particular position in amulti-epitope construct, e.g., adjacent, at the C-terminal side, to theC-terminus of the epitope, encompasses configuring multiple epitopessuch that a desired residue is at a particular position, e.g., adjacentto the epitope, or such that a deleterious residue is not adjacent tothe C-terminus of the epitope. The term also includes inserting an aminoacid residue, preferably a preferred or intermediate amino acid residue,at a particular position. An amino acid residue can also be introducedinto a sequence by substituting one amino acid residue for another.Preferably, such a substitution is made in accordance with analogingprinciples set forth, e.g., in co-pending U.S. Ser. No. 09/260,714 filedMar. 1, 1999 and PCT application number PCT/US00/19774.

The phrases “isolated” or “biologically pure” refer to material that issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state. Thus,isolated peptides in accordance with the invention preferably do notcontain materials normally associated with the peptides in their in situenvironment.

“Link” or “join” refers to any method known in the art for functionallyconnecting peptides, including, without limitation, recombinant fusion,covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding,and electrostatic bonding.

“Major Histocompatibility Complex” or “MHC” is a cluster of genes thatplays a role in control of the cellular interactions responsible forphysiologic immune responses. In humans, the MHC complex is also knownas the HLA complex. For a detailed description of the MHC and HLAcomplexes, see, Paul, FUNDAMENTAL IMMUNOLOGY, 3^(RD) ED., Raven Press,New York, 1993.

As used herein, “middle of the peptide” is a position in a peptide thatis neither amino or carboxyl terminal.

A “minimal number of junctional epitopes” as used herein refers to anumber of junctional epitopes that is lower than what would be createdusing a random selection criterium.

The term “motif” refers to the pattern of residues in a peptide ofdefined length, usually a peptide of from about 8 to about 13 aminoacids for a class I HLA motif and from about 6 to about 25 amino acidsfor a class II HLA motif, which is recognized by a particular HLAmolecule. Peptide motifs are typically different for each proteinencoded by each human HLA allele and differ in the pattern of theprimary and secondary anchor residues.

A “negative binding residue” or “deleterious residue” is an amino acidthat if present at certain positions (typically not a primary anchorposition) in a peptide epitope, results in decreased binding affinity ofthe peptide for the peptide's corresponding HLA molecule.

The phrase “operably linked” refers to a linkage in which a nucleotidesequence is connected to another nucleotide sequence (or sequences) insuch a way as to be capable of altering the functioning of the sequence(or sequences). For example, a nucleic acid or multi-epitope nucleicacid construct that is operably linked to a regulatory sequence, such asa promoter/operator, places expression of the nucleic acid or constructunder the influence or control of the regulatory sequence. Twonucleotide sequences (such as a protein encoding sequence and a promoterregion sequence linked to the 5′ end of the encoding sequence) are saidto be operably linked if induction of promoter function results in thetranscription of the protein encoding sequence mRNA and if the nature ofthe linkage between the two nucleotide sequences does not (1) result inthe introduction of a frame-shift mutation nor (2) prevent theexpression regulatory sequences to direct the expression of the mRNA orprotein. Thus, a promoter region would be operably linked to anucleotide sequence if the promoter were capable of effectingtranscription of that nucleotide sequence.

“Optimizing” refers to increasing the immunogenicity or antigenicity ofa multi-epitope construct having at least one epitope pair by sortingepitopes to minimize the occurrence of junctional epitopes, insertingflanking residues that flank the C-terminus or N-terminus of an epitope,and inserting spacer residue to further prevent the occurrence ofjunctional epitopes or to provide a flanking residue. An increase inimmunogenicity or antigenicity of an optimized multi-epitope constructis measured relative to a multi-epitope construct that has not beenconstructed based on the optimization parameters and is using assaysknown to those of skill in the art, e.g., assessment of immunogenicityin HLA transgenic mice, ELISPOT, inteferon-gamma release assays,tetramer staining, chromium release assays, and presentation ondendritic cells.

The term “peptide” is used interchangeably with “oligopeptide” in thepresent specification to designate a series of residues, typicallyL-amino acids, connected one to the other, typically by peptide bondsbetween the α-amino and carboxyl groups of adjacent amino acids. TheCTL-inducing peptides of the invention are less than about 15 residuesin length, preferably 13 residues or less in length and preferably areabout 8 to about 13 amino acids in length, more preferably about 8 toabout 11 amino acids in length (e.g. 8, 9, 10, or 11), and mostpreferably about 9 amino acids in length. The preferred HTL-inducingoligopeptides are less than about 50 residues in length and usuallyconsist of about 6 to about 30 residues, more usually between about 12to 25, and often about 15 to 20 residues, and can encode an epitopepeptide of about 7 to about 23, preferably about 7 to about 17, morepreferably about 11 to about 15 (e.g. 11, 12, 13, 14, or 15), and mostpreferably about 13 amino acids in length. The multi-epitope constructsdescribed herein preferably include 5 or more, 10 or more, 15 or more,20 or more, or 25 or more epitope nucleic acids.

The nomenclature used to describe peptide, polypeptide, and proteincompounds follows the conventional practice wherein the amino group ispresented to the left (the N-terminus) and the carboxyl group to theright (the C-terminus) of each amino acid residue. When amino acidresidue positions are referred to, they are numbered in an amino tocarboxyl direction with position one being the position closest to theamino terminal end of the epitope, or the peptide, polypeptide orprotein of which it may be a part. In the formulae representing selectedspecific embodiments of the present invention, the amino- andcarboxyl-terminal groups, although not specifically shown, are in theform they would assume at physiologic pH values, unless otherwisespecified. In the amino acid structure formulae, each residue isgenerally represented by standard three-letter or single-letterdesignations. The L-form of an amino acid residue is represented by acapital single letter or a capital first letter of a three-lettersymbol, and the D-form for those amino acids having D-forms isrepresented by a lower case single letter or a lower case three lettersymbol. Glycine has no asymmetric carbon atom and is simply referred toas “Gly” or G. Symbols for the amino acids are shown below.

Single Letter Symbol Three Letter Symbol Amino Acids A Ala Alanine C CysCysteine D Asp Aspartic Acid E Glu Glutamic Acid F Phe Phenylalanine GGly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu LeucineM Met Methionine N Asn Asparagine P Pro Proline Q Gln Glutamine R ArgArginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan YTyr Tyrosine

Amino acid “chemical characteristics” are defined as: Aromatic (F, W,Y); Aliphatic-hydrophobic (L, I, V, M); Small polar (S, T, C); Largepolar (Q, N); Acidic (D, E); Basic (R, H, K); Proline; Alanine; andGlycine.

The terms “PanDR binding peptide,” “PanDR binding epitope,” “PADRE®peptide,” and “PADRE® epitope,” refer to a type of HTL peptide which isa member of a family of molecules that binds more than one HLA class IIDR molecule. PADRE® peptides bind to most HLA-DR molecules and stimulatein vitro and in vivo human helper T lymphocyte (HTL) responses. Thepattern that defines the PADRE® family of molecules can be thought of asan HLA Class II supermotif. For example, a PADRE® peptide may comprisethe formula: aKXVAAWTLKAAa (SEQ ID NO:1), where “X” is eithercyclohexylalanine, phenylalanine or tyrosine, and “a” is eitherD-alanine or L-alanine, has been found to bind to most HLA-DR alleles,and to stimulate the response of T helper lymphocytes from mostindividuals, regardless of their HLA type. An alternative of a PADRE®epitope comprises all “L” natural amino acids which can be provided inpeptide/polypeptide form and in the form of nucleic acids that encodethe epitope, e.g., in multi-epitope constructs. Specific examples ofPADRE® peptides are also disclosed herein.

“Pharmaceutically acceptable” refers to a generally non-toxic, inert,and/or physiologically compatible composition.

“Presented to an HLA Class I processing pathway” means that themulti-epitope constructs are introduced into a cell such that they arelargely processed by an HLA Class I processing pathway. Typically,multi-epitope constructs are introduced into the cells using expressionvectors that encode the multi-epitope constructs. HLA Class II epitopesthat are encoded by such a multi-epitope construct are also presented onClass II molecules, although the mechanism of entry of the epitopes intothe Class II processing pathway is not defined.

A “primary anchor residue” or a “primary MHC anchor” is an amino acid ata specific position along a peptide sequence that is understood toprovide a contact point between the immunogenic peptide and the HLAmolecule. One to three, usually two, primary anchor residues within apeptide of defined length generally defines a “motif” for an immunogenicpeptide. These residues are understood to fit in close contact withpeptide binding grooves of an HLA molecule, with their side chainsburied in specific pockets of the binding grooves themselves. In oneembodiment, for example, the primary anchor residues of an HLA class Iepitope are located at position 2 (from the amino terminal position) andat the carboxyl terminal position of a 9-residue peptide epitope inaccordance with the invention. The primary anchor positions for eachmotif and supermotif are described, for example, in Tables I and III ofPCT/US00/27766, or PCT/US00/19774. Preferred amino acids that can serveas in the anchors for most Class II epitopes consist of M and F inposition one and V, M, S, T, A and C in position six. Tolerated aminoacids that can occupy these positions for most Class II epitopes consistof L, I, V, W, and Y in position one and P, L and I in position six. Thepresence of these amino acids in positions one and six in Class IIepitopes defines the HLA-DR1, 4, 7 supermotif. The HLA-DR3 binding motifis defined by preferred amino acids from the group of L, I, V, M, F, Yand A in position one and D, E, N, Q, S and T in position four and K, Rand H in position six. Other amino acids may be tolerated in thesepositions but they are not preferred.

Furthermore, analog peptides can be created by altering the presence orabsence of, i.e. replacing, a particular residue in these primary anchorpositions. Such analogs are used to modulate the binding affinity of apeptide comprising a particular motif or supermotif.

“Promiscuous recognition” occurs where a distinct peptide is recognizedby the same T cell clone in the context of various HLA molecules.Promiscuous recognition or binding is synonymous with cross-reactivebinding.

A “protective immune response” or “therapeutic immune response” refersto a CTL and/or an HTL response to an antigen derived from an infectiousagent or a tumor antigen, which in some way prevents or at leastpartially arrests disease symptoms, side effects or progression. Theimmune response may also include an antibody response that has beenfacilitated by the stimulation of helper T cells.

By “regulatory sequence” is meant a polynucleotide sequence thatcontributes to or is necessary for the expression of an operablyassociated nucleic acid or nucleic acid construct in a particular hostorganism. The regulatory sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aninternal ribosome binding site (IRES). Eukaryotic cells are known toutilize promoters, polyadenylation signals, and enhancers. Promoter maybe a CMV promoter or other promoter described herein or known in theart. Regulatory sequences include IRESs. Other specific examples ofregulatory sequences are described herein and otherwise known in theart.

The term “residue” refers to an amino acid or amino acid mimeticincorporated into a peptide or protein by an amide bond or amide bondmimetic.

A “secondary anchor residue” is an amino acid at a position other than aprimary anchor position in a peptide that may influence peptide binding.A secondary anchor residue occurs at a significantly higher frequencyamongst bound peptides than would be expected by random distribution ofamino acids at one position. The secondary anchor residues are said tooccur at “secondary anchor positions.” A secondary anchor residue can beidentified as a residue that is present at a higher frequency among highor intermediate affinity binding peptides, or a residue otherwiseassociated with high or intermediate affinity binding. For example,analog peptides can be created by altering the presence or absence of,i.e. replacing, a particular residue in these secondary anchorpositions. Such analogs are used to finely modulate the binding affinityof a peptide comprising a particular motif or supermotif. Theterminology “fixed peptide” is sometimes used to refer to an analogpeptide.

“Sorting epitopes” refers to determining or designing an order of theepitopes in a multi-epitope construct.

A “spacer” refers to a sequence that is inserted between two epitopes ina multi-epitope construct to prevent the occurrence of junctionalepitopes and/or to increase the efficiency of processing. Amulti-epitope construct may have one or more spacer nucleic acids. Aspacer nucleic acid may flank each epitope nucleic acid in a construct,or the spacer nucleic acid to epitope nucleic acid ratio may be about 2to 10, about 5 to 10, about 6 to 10, about 7 to 10, about 8 to 10, orabout 9 to 10, where a ratio of about 8 to 10 has been determined toyield favorable results for some constructs.

The spacer nucleic acid may encode one or more amino acids. A spacernucleic acid flanking a class I HLA epitope in a multi-epitope constructis preferably between one and about eight amino acids in length. Aspacer nucleic acid flanking a class II HLA epitope in a multi-epitopeconstruct is preferably greater than five, six, seven, or more aminoacids in length, and more preferably five or six amino acids in length.

The number of spacers in a construct, the number of amino acids in aspacer, and the amino acid composition of a spacer can be selected tooptimize epitope processing and/or minimize junctional epitopes. It ispreferred that spacers are selected by concomitantly optimizing epitopeprocessing and junctional motifs. Suitable amino acids for optimizingepitope processing are described herein. Also, the suitable amino acidspacing for minimizing the number of junctional epitopes in a constructis described herein for class I and class II HLAs. For example, spacersflanking class II HLA epitopes preferably include G, P, and/or Nresidues as these are not generally known to be primary anchor residues(see, e.g., PCT/US00/19774). A particularly preferred spacer forflanking a class II HLA epitope includes alternating G and P residues,for example, (GP)_(n), (PG)_(n), (GP)_(n)G, (PG)_(n)P, and so forth,where n is an integer between one and ten, preferably two or about two,and where a specific example of such a spacer is GPGPG (SEQ ID NO:2). Apreferred spacer, particularly for class I HLA epitopes, comprises one,two, three or more consecutive alanine (A) residues (see, for example,FIG. 23A, which depicts a spacer having three consecutive alanineresidues).

In some multi-epitope constructs, it is sufficient that each spacernucleic acid encodes the same amino acid sequence. In multi-epitopeconstructs having two spacer nucleic acids encoding the same amino acidsequence, the spacer nucleic acids encoding those spacers may have thesame or different nucleotide sequences, where different nucleotidesequences may be preferred to decrease the likelihood of unintendedrecombination events when the multi-epitope construct is inserted intocells.

In other multi-epitope constructs, one or more of the spacer nucleicacids may encode different amino acid sequences. While many of thespacer nucleic acids may encode the same amino acid sequence in amulti-epitope construct, one, two, three, four, five or more spacernucleic acids may encode different amino acid sequences, and it ispossible that all of the spacer nucleic acids in a multi-epitopeconstruct encode different amino acid sequences. Spacer nucleic acidsmay be optimized with respect to the epitope nucleic acids they flank bydetermining whether a spacer sequence will maximize epitope processingand/or minimize junctional epitopes, as described herein.

Multi-epitope constructs may be distinguished from one another accordingto whether the spacers in one construct optimize epitope processing orminimize junctional epitopes over another construct, and preferably,constructs may be distinguished where one construct is concomitantlyoptimized for epitope processing and junctional epitopes over the other.Computer assisted methods and in vitro and in vivo laboratory methodsfor determining whether a construct is optimized for epitope processingand junctional motifs are described herein.

A “subdominant epitope” is an epitope that evokes little or no responseupon immunization with whole antigens which comprise the epitope, butfor which a response can be obtained by immunization with an isolatedepitope, and this response (unlike the case of cryptic epitopes) isdetected when whole protein is used to recall the response in vitro orin vivo.

A “supermotif” is an amino acid sequence for a peptide that providesbinding specificity shared by HLA molecules encoded by two or more HLAalleles. Preferably, a supermotif-bearing peptide is recognized withhigh or intermediate affinity (as defined herein) by two or more HLAantigens.

“Synthetic peptide” refers to a peptide that is not naturally occurring,but is man-made using such methods as chemical synthesis or recombinantDNA technology.

A “TCR contact residue” or “T cell receptor contact residue” is an aminoacid residue in an epitope that is understood to be bound by a T cellreceptor; these are defined herein as not being a primary MHC anchor. Tcell receptor contact residues are defined as the position/positions inthe peptide where all analogs tested induce T-cell recognition relativeto that induced with a wildtype peptide.

The term “homology,” as used herein, refers to a degree ofcomplementarity between two nucleotide sequences. The word “identity”may substitute for the word “homology” when a nucleic acid has the samenucleotide sequence as another nucleic acid. Sequence homology andsequence identity can also be determined by hybridization studies underhigh stringency and/or low stringency, and disclosed herein are nucleicacids that hybridize to the multi-epitope constructs under lowstringency or under high stringency. Also, sequence homology andsequence identity can be determined by analyzing sequences usingalgorithms and computer programs known in the art. Such methods be usedto assess whether a nucleic acid is identical or homologous to themulti-epitope constructs disclosed herein. The invention pertains inpart to nucleotide sequences having 80% or more, 85% or more, 90% ormore, 95% or more, 97% or more, 98% or more, or 99% or more identity tothe nucleotide sequence of a multi-epitope construct disclosed herein.

As used herein, the term “stringent conditions” refers to conditionsthat permit hybridization between nucleotide sequences and thenucleotide sequences of the disclosed multi-epitope constructs. Suitablestringent conditions can be defined by, for example, the concentrationsof salt or formamide in the prehybridization and hybridizationsolutions, or by the hybridization temperature, and are well known inthe art. In particular, stringency can be increased by: reducing theconcentration of salt, increasing the concentration of formamide, orraising the hybridization temperature. For example, hybridization underhigh stringency conditions could occur in about 50% formamide at about37° C. to 42° C. In particular, hybridization could occur under highstringency conditions at 42° C. in 50% formamide, 5×SSPE, 0.3% SDS, and200 μg/ml sheared and denatured salmon sperm DNA or at 42° C. in asolution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA,followed by washing the filters in 0.1×SSC at about 65° C. Hybridizationcould occur under reduced stringency conditions in about 35% to 25%formamide at about 30° C. to 35° C. For example, reduced stringencyconditions could occur at 35° C. in 35% formamide, 5×SSPE, 0.3% SDS, and200 μg/ml sheared and denatured salmon sperm DNA. The temperature rangecorresponding to a particular level of stringency can be furthernarrowed by calculating the purine:pyrimidine ratio of the nucleic acidof interest, and adjusting the temperature accordingly. Variations onthe above ranges and conditions are well known in the art.

In addition to utilizing hybridization studies to assess sequenceidentity or sequence homology, known computer programs may be used todetermine whether a particular nucleic acid is homologous to amulti-epitope construct disclosed herein. An example of such a programis the Bestfit program (Wisconsin Sequence Analysis Package, Version 8for Unix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711), and other sequence alignment programs areknown in the art and may be utilized for determining whether two or morenucleotide sequences are homologous. Bestfit uses the local homologyalgorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between twosequences. When using Bestfit or any other sequence alignment program todetermine whether a particular sequence is, for instance, 95% identicalto a reference sequence, the parameters may be set such that thepercentage of identity is calculated over the full length of thereference nucleotide sequence and that gaps in homology of up to 5% ofthe total number of nucleotides in the reference sequence are allowed.

Acronyms used herein are as follows:

-   APC: Antigen presenting cell-   CD3: Pan T cell marker-   CD4: Helper T lymphocyte marker-   CD8: Cytotoxic T lymphocyte marker-   CEA: Carcinoembryonic antigen-   CFA: Complete Freund's Adjuvant-   CMV: Human Cytomegalovirus-   CTL: Cytotoxic T lymphocytes-   Cardiotoxin: A natural 60 amino acid peptide that causes local    muscle destruction (a protein kinase C inhibitor)-   DC: Dendritic cells. DC functioned as potent antigen presenting    cells by stimulating cytokine release from CTL lines that were    specific for a model peptide derived from hepatitis B virus (HBV).    In vitro experiments using DC pulsed ex vivo with an HBV peptide    epitope have stimulated CTL immune responses in vitro following    delivery to naïve mice.-   DMSO: Dimethylsulfoxide-   DNA: Deoxyribonucleic acid-   EBV: Epstein Barr Virus-   ELISA: Enzyme-linked immunosorbant assay-   ELISPOT: ELISA-like procedure that detects individual cells    secreting probed cytokine as a distinct spot on a culture membrane-   Epigene: Multi-epitope DNA constructs-   E:T: Effector:target ratio-   FACS: Flourescence-activated cell sorter-   FCS: Fetal calf serum-   G-CSF: Granulocyte colony-stimulating factor-   GM-CSF: Granulocyte-macrophage (monocyte)-colony stimulating factor-   HBV: Hepatitis B virus-   HER2/Neu: c-erbB-2-   HIV: Human Immunodeficiency Virus-   HLA: Human leukocyte antigen-   HLA-DR: Human leukocyte antigen class II-   HPLC: High Performance Liquid Chromatography-   HTC: Helper T cells-   HTL: Helper T Lymphocyte-   ID: Identity-   IFA: Incomplete Freund's Adjuvant-   IFNγ: Interferon gamma-   IL-4: Interleukin-4 cytokine-   IRES: Internal ribosome entry site-   IV: Intravenous-   LU_(30%): Cytotoxic activity required to achieve 30% lysis at a    100:1 (E:T) ratio-   MAb: Monoclonal antibody-   MAGE: Melanoma antigen-   MHC: Major Histocompatibility Complex-   MLR: Mixed lymphocyte reaction-   MNC: Mononuclear cells-   PADRE™: a PanDR binding peptide-   PATR: Pan Troglodytes-   PB: Peripheral blood-   PBL: Peripheral blood lymphocyte-   PBMC: Peripheral blood mononuclear cell-   SC: Subcutaneous-   SDS: Sodium dodecyl sulfate-   S.E.M.: Standard error of the mean-   SU: Secretory units-   QD: Once a day dosing-   TAA: Tumor associated antigen-   TCR: T cell receptor-   TNF: Tumor necrosis factor-   WBC: White blood cells

This application may be relevant to U.S. Ser. No. 09/189,702 filed Nov.10, 1998, which is a CIP of U.S. Ser. No. 08/205,713 filed Mar. 4, 1994,which is a CIP of U.S. Ser. No. 08/159,184 filed Nov. 29, 1993 and nowabandoned, which is a CIP of U.S. Ser. No. 08/073,205 filed Jun. 4, 1993and now abandoned, which is a CIP of U.S. Ser. No. 08/027,146 filed Mar.5, 1993 and now abandoned. The present application is also related toU.S. Ser. No. 09/226,775, which is a CIP of U.S. Ser. No. 08/815,396,which claims the benefit of U.S. Ser. No. 60/013,113, now abandoned.Furthermore, the present application is related to U.S. Ser. No.09/017,735, which is a CIP of abandoned U.S. Ser. No. 08/589,108; U.S.Ser. No. 08/753,622, U.S. Ser. No. 08/822,382, abandoned U.S. Ser. No.60/013,980, U.S. Ser. No. 08/454,033, U.S. Ser. No. 09/116,424, and U.S.Ser. No. 08/349,177. The present application is also related to U.S.Ser. No. 09/017,524, U.S. Ser. No. 08/821,739, abandoned U.S. Ser. No.60/013,833, U.S. Ser. No. 08/758,409, U.S. Ser. No. 08/589,107, U.S.Ser. No. 08/451,913, U.S. Ser. No. 08/186,266, U.S. Ser. No. 09/116,061,and U.S. Ser. No. 08/347,610, which is a CIP of U.S. Ser. No.08/159,339, which is a CIP of abandoned U.S. Ser. No. 08/103,396, whichis a CIP of abandoned U.S. Ser. No. 08/027,746, which is a CIP ofabandoned U.S. Ser. No. 07/926,666. The present application may also berelevant to U.S. Ser. No. 09/017,743, U.S. Ser. No. 08/753,615; U.S.Ser. No. 08/590,298, U.S. Ser. No. 09/115,400, and U.S. Ser. No.08/452,843, which is a CIP of U.S. Ser. No. 08/344,824, which is a CIPof abandoned U.S. Ser. No. 08/278,634. The present application may alsobe related to provisional U.S. Ser. No. 60/087,192 and U.S. Ser. No.09/009,953, which is a CIP of abandoned U.S. Ser. No. 60/036,713 andabandoned U.S. Ser. No. 60/037,432. In addition, the present applicationmay be relevant to U.S. Ser. No. 09/098,584, and U.S. Ser. No.09/239,043. The present application may also be relevant to co-pendingU.S. Ser. No. 09/583,200 filed May 30, 2000, U.S. Ser. No. 09/260,714filed Mar. 1, 1999, and U.S. Provisional Application No. 60/239,008,filed Oct. 6, 2000, and U.S. Provisional Application No. 60/166,529,filed Nov. 18, 1999. In addition, the present application may also berelevant to U.S. Provisional Application No. 60/239,008, filed Oct. 6,2000, now abandoned; co-pending U.S. application Ser. No. 10/130,548,which is the U.S. Natl. Phase Application of PCT/US00/31856, filed Nov.20, 2000 and published as WO 01/36452 on May 25, 2001; and co-pendingU.S. application Ser. No. 10/116,118, filed Apr. 5, 2002. All of theabove applications are incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates data on three different multi-epitope constructs,incorporating 20 to 25 different CTL epitopes each.

FIG. 2 illustrates two different synthetic polypeptides (FIG. 2 a) wherethe first construct incorporates four different epitopes linearlycosynthetized, and the second construct incorporates a GPGPG (SEQ IDNO:2) spacer. FIG. 2 b illustrates the capacity of 2 nanomoles of thesedifferent constructs to prime for proliferative responses to the variousepitopes in IA^(b) positive mice, compared to the responses induced byequimolar amounts of a pool of the same peptides (3 micrograms of eachpeptide).

FIG. 3 depicts the structure of multi-epitope DNA constructs. The HLArestriction is shown above each epitope, the A*0201 epitopes are bolded.The HLA binding affinity (IC₅₀ nM) is provided below each epitope. (a)Schematic of HIV-FT illustrating order of the encoded epitopes. (b)Schematics of the of the HBV-specific constructs. The C+1 amino acidrelative to Core 18 is indicated with an arrow. The HBV-specificconstructs with single amino acid insertions at the C₁ position of Core18 are illustrated as HBV.1X.

FIG. 4 illustrates the immunogenicity of the HLA-A*0201 epitopes inHIV-FT in HLA-A*0201/K^(b) transgenic mice. (a) Representative CTLresponses against epitopes Pol 498 (circles), Vpr 62 (triangle), Gag 386(squares). Cytotoxicity was assayed in a ⁵¹Cr release assay againstJurkat-HLA-A*0201/K^(b) target cells in the presence (filled symbols) orabsence (open symbols) of each peptide. (b) Summary of CTL responses ofimmunogenicity of HIV-FT in HLA-A*0201/K^(b) transgenic mice. Barsindicate the geometric mean CTL response of positive cultures. Thefrequency of positive CTL cultures is also indicated.

FIG. 5 shows the influence of the C+1 amino acid on epitopeimmunogenicity. A database incorporating CTL responses from a variety ofmulti-epitope constructs representing 94 epitope/C+1 amino acidcombinations was analyzed to determine the frequency (%) of instances inwhich a particular combination was associated with an optimal CTLresponse. CTL responses were considered optimal if greater than 100 SUor 20 LU in at least 30% of the cultures measured. The number of times agiven epitope/C+1 amino acid combination was observed is also provided.

FIG. 6 shows CTL responses to HBV-specific constructs (a) CTL responsesto Core 18 epitope following DNA immunization of HLA-A*0201/K^(b)transgenic mice. (b) CTL responses to HBV Core 18 following DNAimmunization of HLA-A*0201/K^(b) transgenic mice with constructs whichvary by a single amino acid insertion at the C+1 position of Core 18.

FIG. 7 shows levels of HBV Core 18 presentation in HBV.1 (shaded bars)and HBV.1K (hatched bars) transfected cell lines. Epitope presentationwas quantified using peptide-specific CTL lines. Presentation of HBV Pol455 is shown for comparative purposes.

FIG. 8 depicts data for 221A2 K^(b) target cells transfected with theHIV-FT epigene construct. These transfected cells were assayed for theircapacity to present epitopes to CTL lines derived from HLA transgenicmice and specific for various HIV-derived CTL epitopes. To correct fordifferences in antigen sensitivity of different CTL lines, peptide dosetitrations, using untransfected cells as APC, were run in parallel.

FIG. 9 shows HIV multi-epitope constructs optimized using the methods ofthe present invention

FIG. 10 illustrates a computer system for performing automaticoptimization of multi-epitope constructs in accordance with oneembodiment of the invention.

FIGS. 11A-B illustrate an exemplary input text file containing userinput parameters used for executing a Junctional Analyzer program, inaccordance with one embodiment of the invention.

FIG. 12 illustrates a flow chart diagram of a software program foridentifying optimal multi-epitope constructs, in accordance with oneembodiment of the invention.

FIGS. 13A-D illustrate an exemplary output text file containing outputresults of a Junctional Analyzer program, in accordance with oneembodiment of the invention.

FIG. 14A depicts CTL responses induced by EP-HIV-90 relative toindividual peptides in IFA, and FIG. 14B depicts CTL responses inducedby PfCTL.1, PfCTL.2, and PfCTL.3 relative to individual peptides.

FIG. 15 shows the effect of GPGPG (SEQ ID NO:2) spacers in class IIepitope constructs HIV 75mer and HIV 60mer on HTL responses toparticular epitopes.

FIG. 16 depicts HTL responses to particular epitopes present in theEP-HIV-1043-PADRE® construct.

FIG. 17 is a schematic depicting the epitopes present in HIV 75mer,EP-HIV-1043, and the EP-HIV-1043-PADRE® construct.

FIGS. 18A-N show the amino acid sequences and nucleic acid sequences ofcertain multi-epitope constructs.

-   -   FIGS. 19 A-E show the amino acid sequences and nucleic acid        sequences of certain multi-epitope constructs.

FIGS. 20A-20F show the HBV CTL epitopes used to construct three relatedepigene constructs, HBV-2, HBV-2A and HBV-2B, the order of epitopes inthe epigene constructs, the immune responses induced in HLA-A2 orHLA-A3/11 transgenic mice and the amino acid and nucleic acid sequencesof the epigene constructs. In FIG. 20B, the signal sequence in HBV-2,HBV-2A and HBV-2B is the Ig kappa consensus signal sequence, althoughother signal sequences could be utilized.

FIGS. 21A-21E show the HBV CTL epitopes used to construct two 21 CTLepitope epigene constructs, HBV-21A and HBV-21B, the order of epitopesin the epigene constructs, the immune responses induced in HLA-A2 orHLA-A3/11 transgenic mice and the amino acid and nucleic acid sequencesof the epigene constructs.

FIGS. 22A-22E show the HBV CTL epitopes used to construct two 30 CTLepitope epigene constructs, HBV-30B and HBV-30C, the order of epitopesin the epigene constructs, the immune responses induced in HLA-A2 orHLA-A3/11 transgenic mice and the amino acid and nucleic acid sequencesof the epigene constructs.

FIGS. 23A-23C show the modifications made to spacers flanking two HLA-A2restricted CTL epitopes in the HBV-30C epigene construct. Modificationswere designed to increase the efficiency of processing and subsequentpresentation and thus, increase immunogenicity of the epitopes.Immunogenicity was measured using HLA-A2 or HLA-A3/11 transgenic mice,and the amino acid and nucleic acid sequences of the epigene constructare noted. In FIG. 23A, the lysine (K) spacer flanking the Core 18epitope in HBV-30C were modified to include three alanine residues (AAA)in HBV-30CL. Also, one asparagine (N) spacer flanking env 183 epitope inHBV-30C was modified to include three alanine residues (AAA) inHBV-30CL.

FIGS. 24A-24C show HTL epitopes, and their binding affinity to selectedHLA-DR alleles, used to construct a multi-epitope vaccine comprising HTLepitopes separated by GPGPG (SEQ ID NO:2) amino acid spacers. Thenucleic acid sequence of the multi-epitope vaccine and the amino acidsequence encoded by the nucleic acid are shown in FIG. 24C.

FIGS. 25A-B show the population coverage for CTL epitopes contained inGCR-5835. FIG. 25A. Percentage of individuals projected to present theindicated number of HLA-A/B-epitope combinations in a compositepopulation derived from gene frequencies in Asian, Black, EuropeanCaucasian, and North American Caucasian populations (Black bars). Alsoshown on the right axis is the cumulative plot of percent populationcoverage (Open circles). FIG. 25B. Summary of the cumulative percentprojected population coverage in Asian, Black, European Caucasian, andNorth American Caucasian populations as a function of the number ofepitopes bound by HLA alleles.

FIGS. 26A-26B show population coverages for epitopes contained in alist. FIG. 26A. Percentage of individuals projected to present theindicated number of HLA-DR-epitope combinations in a compositepopulation derived from gene frequencies in Asian, Black, EuropeanCaucasian, and North American Caucasian populations (black bars). Alsoshown on the right axis is the cumulative plot of percent populationcoverage (open circles). FIG. 26B. Summary of the cumulative percentprojected population coverage in Asian, Black, European Caucasian, andNorth American Caucasian populations as a function of the number ofepitopes bound by HLA alleles.

FIGS. 27A-27B show (A) a schematic of HBV30K and (B) the HLA supertyperestriction of the component epitopes. Immunogenicity of a vaccine 30epitope epigene construct. HLA-A2 or -A11 transgenic mice were immunizedintramuscularly with 100 μg of the vaccine HBV epigene plasmid HBV30K orthe prototype HBV vaccine HBV2. Eleven days after the immunizationsplenocytes were stimulated in vitro with the epitopes encoded in thevaccine. After six days in culture the epitope-specific CTL responseswere measured using an in situ IFN-γ ELISA assay.

FIGS. 28A-28B show a schematic of the HBV HTL vaccine construct and itsimmunogenicity. FIG. 26A. GPGPG (SEQ ID NO:2) spacers introduced betweenepitopes are indicated. FIG. 28B. H2^(bxd) mice were immunizedintramuscularly with 100 μg of a vaccine HBV HTL epigene construct orthe individual peptides emulsified in CFA. Eleven days after theimmunization CD4 T cells were purified from splenocytes and HTLresponses were measured using a primary IFN-γ ELISPOT assay.

FIGS. 29A-29B show HBV vaccine plasmid configurations and their relativeimmunogenicity. FIG. 29A. Schematic (i) dual CMV promoter plasmid; (ii)IRES containing plasmid; (iii) CTL+HTL epigene construct fusion. FIG.29B. Relative immunogenicity of different vaccine configurations.HLA-A2-transgenic mice were immunized intramuscularly with 100 μg ofHBV30K (CTL epigene construct control), HBV30K.H1 (dual CMV promoterplasmid), HBV30K.H3 (IRES containing plasmid) or HBV30K/HTL (GCR-5835;CTL+HTL epigene construct fusion). Eleven days after the immunizationsplenocytes were stimulated in vitro with the epitopes encoded in thevaccine. After six days in culture the epitope-specific CTL responseswere measured using an in situ IFN-γ ELISA assay.

FIG. 30 shows the relative immunogenicity of GCR-5835 and GCR-3697.HLA-A2 transgenic mice were immunized intramuscularly with either 50 μgor 5 μg of the GCR-5835 or GCR-3697. Eleven days after the immunizationCD8+ cells were isolated from splenocytes and epitope-specific CTLresponses were measured using an IFN-γ ELISPOT assay.

FIG. 31 shows a comparison of PVP formulated, naked, and CT GCR-5835.HLA-A2. 1/K^(b) transgenic mice were immunized a single time with 100 μgof GCR 5835 formulated in PVP, naked, or naked in cardiotoxin (CT)pre-treated animals. After eleven days in vivo, splenocytes wererestimulated in vitro with the indicated peptides. After six days, IFN-γwas measured in response to peptide in an in situ ELISA assay. Data arepresented as the geometric mean of the secretory units (SU) for positivecultures, x/, standard deviation. The frequency of positivecultures/total cultures tested is indicated above each bar.

FIG. 32 shows a comparison of GCR-5835 and the lipopeptide vaccine.HLA-A2.1/K^(b) transgenic mice were immunized with either 100 μg ofGCR-5835 in cardiotoxin (CT) pre-treated animals or 100 μg of thelipopeptide vaccine. After eleven days in vivo, CD8+ splenocytes wereisolated, and IFN-γ was measured in response to the indicated peptide inan ELISPOT assay (A). Data are presented as the average spot formingcells (SFC) per 10⁶ splenocytes plated. Alternatively, splenocytes wererestimulated in vitro with the indicated peptides. After six days, IFN-γwas measure in response to peptide in an in situ ELISA assay (B). Dataare presented as the geometric mean of the secretory units (SU) forpositive cultures, ± standard deviation.

FIGS. 33A-33B show a summary of immunogenicity data from individualmice. HLA-A2.1/K^(b) transgenic mice were either not immunized, orimmunized with 100 μg of PVP-formulated GCR-5835 in a singleimmunization (A), or immunized twice at a 7 day interval (B). Elevendays after the final immunization, splenocytes from each mouse wererestimulated in vitro with a pool of the indicated peptides. After sixdays, IFN-γ was measured in response to the individual peptides as wellas a pool of all peptides in an ELISPOT assay. Data are presented as theaverage spot forming cells (SFC) per 10⁶ splenocytes plated.

FIG. 34 shows a schematic of the HBV AOSIb and HBV AOSIb2 constructs.The HBV AOSIb2 construct has additional amino acids added (indicatedwith arrows above the schematic) to enhance proteasomal processing whilethe HBV AOSIb construct has no added residues.

FIGS. 35A-35E show the results after transient transfection of human 293cells in the presence or absence of the proteasome inhibitor MG132. Theproteasome inhibitor MG132 was added at 5 μM 24 hours post-transfection.Flourescence in live cells was detected by flow cytometry andflourescence microscopy 24 hours after addition of the proteasomeinhibitor (unless otherwise noted). (A) Flow cytometry (FACS) resultsfor a time-course of cells transfected with plasmid AOSIb. (B) Flowcytometry (FACS) results at 24 hours for cells transfected with plasmidHBV AOSIb. (C) Flow cytometry (FACS) results at 24 hours for cellstransfected with plasmid HBV AOSIb2. (D) Data are presented graphicallyas a comparison of fluorescence intensity. (E) The relative increase influoresence intensity is compared between control plasmid, HBV AOSIb,and HBV AOSIb2 for the above experiments.

FIG. 36 shows the amount of proteins detectable upon addition of theproteasome inhibitors lactacystin (25 uM) or MG132 (5 uM). Whole celllysates were prepared from transfected cells and transferred to ablotting membrane. Proteins were detected using an antibody against thefusion partner protein. Arrows indicate the predicted size of thefull-length fusion proteins.

FIGS. 37A-37B show epitope-specific T cell responses measured in HLAtransgenic mice immunized with GCR-3697 using splenic lymphocytesobtained 11-14 days following immunization. Groups of 6-9 HLA-transgenicmice were immunized bilaterally with 100 μg of DNA in the tibialisanterior muscle. DNA was delivered in either PBS or PVP formulations; inthe case of PBS formulations the injection site was pre-treated bycardiotoxin injection.

FIG. 38 shows a comparison of fluorescence intensity measured by FACSanalysis for the 3 plasmids: no epitope construct (flourescent proteinonly), fluorescent conjugated polyepitope HBV AOSIb, or fluorescentconjugated polyepitope HBV AOSIb2. Human 293 cells were transfected withplasmid and the proteasome inhibitor MG132 was added at 5 μM 24 hourspost-transfection. Flourescence in live cells was detected by FACS 24hours after addition of the proteasome inhibitor.

FIG. 39 shows fluorescence microscopy images for cells cultured with: noepitope construct (fluorescent protein only), fluorescent conjugatedpolyepitope HBV AOSIb, or fluorescent conjugated polyepitope HBV AOSIb2.Human 293 cells were transfected with plasmid and the proteasomeinhibitor MG132 was added at 5 μM 24 hours post-transfection.Flourescence in live cells was detected by fluoresence microscopy 24hours after addition of the proteasome inhibitor.

DETAILED DESCRIPTION OF THE INVENTION

The invention is described in detail below with reference to the figureswherein like elements are referenced with like numerals throughout.

The invention provides a method and system for optimizing the efficacyof multi-epitope vaccines, preferably to minimize the number ofjunctional epitopes and maximize, or at least increase, theimmunogenicity and/or antigenicity of multi-epitope vaccines. Thepresent invention also provides multi-epitope nucleic acid constructsencoding a plurality of CTL and/or HTL epitopes and polypeptides encodedby such constructs, as well as cells comprising such constructs and/orpolypeptides, compositions comprising such constructs, polypeptides,and/or cells, and methods for stimulating an immune response (e.g.therapeutic methods) utilizing such constructs and/or polypeptides andcells.

In one embodiment of the invention, a computerized method for designinga multi-epitope construct having multiple epitopes includes the stepsof: storing a plurality of input parameters in a memory of a computersystem, the input parameters including a plurality of epitopes, at leastone motif for identifying junctional epitopes, a plurality of amino acidinsertions and at least one enhancement weight value for each insertion;generating a list of epitope pairs from the plurality of epitopes;determining for each epitope pair at least one optimum combination ofamino acid insertions based on the at least one motif, the plurality ofinsertions and the at least one enhancement weight value for eachinsertion; and identifying at least one optimum arrangement of theplurality of epitopes, wherein a respective one of the at least oneoptimum combination of amino acid insertions is inserted at a respectivejunction of two epitopes, so as to provide an optimized multi-epitopeconstruct. In a preferred embodiment, the step of identifying at leastone optimum arrangement of epitopes may be accomplished by performingeither an exhaustive search wherein all permutations of arrangements ofthe plurality of epitopes are evaluated or a stochastic search whereinonly a subset of all permutations of arrangements of the plurality ofepitopes are evaluated.

In a further embodiment, the method determines for each epitope pair atleast one optimum combination of amino acid insertions by calculating afunction value (F) for each possible combination of insertions for eachepitope pair, wherein the number of insertions in a combination mayrange from 0 to a maximum number of insertions (MaxInsertions) valueinput by a user, and the function value is calculated in accordance withthe equation F═(C+N)/J, when J>0, and F=2(C+N), when J=0, wherein Cequals the enhancement weight value of a C+1 flanking amino acid, Nequals the enhancement weight value of an N−1 flanking amino acid, and Jequals the number of junctional epitopes detected for each respectivecombination of insertions in an epitope pair based on said at least onemotif.

In another embodiment of the invention, a computer system for designinga multi-epitope construct having multiple epitopes, includes: a memoryfor storing a plurality of input parameters such as a plurality ofepitopes, at least one motif for identifying junctional epitopes, aplurality of amino acid insertions and at least one enhancement weightvalue for each insertion; a processor for retrieving the inputparameters from memory and generating a list of epitope pairs from theplurality of epitopes; wherein the processor further determines for eachepitope pair at least one optimum combination of amino acid insertions,based on the at least one motif, the plurality of insertions and the atleast one enhancement weight value for each insertion. The processorfurther identifies at least one optimum arrangement of the plurality ofepitopes, wherein a respective one of the optimum combinations of aminoacid insertions are inserted at a respective junction of two epitopes,to provide an optimized multi-epitope construct; and a display monitor,coupled to the processor, for displaying at least one optimumarrangement of the plurality of epitopes to a user.

In a further embodiment, the invention provides a data storage devicestoring a computer program for designing a multi-epitope constructhaving multiple epitopes, the computer program, when executed by acomputer system, performing a process that includes the steps of:retrieving a plurality of input parameters from a memory of a computersystem, the input parameters including, for example, a plurality ofepitopes, at least one motif for identifying junctional epitopes, aplurality of amino acid insertions and at least one enhancement weightvalue for each insertion; generating a list of epitope pairs from theplurality of epitopes; determining for each epitope pair at least oneoptimum combination of amino acid insertions based on the at least onemotif, the plurality of insertions and the at least one enhancementweight value for each insertion; and identifying at least one optimumarrangement of the plurality of epitopes, wherein a respective one ofthe at least one optimum combination of amino acid insertions isinserted at a respective junction of two epitopes, so as to provide anoptimized multi-epitope construct.

In another embodiment, the invention provides a method and system fordesigning a multi-epitope construct that comprises multiple epitopes.The method comprising steps of: (i) sorting the multiple epitopes tominimize the number of junctional epitopes; (ii) introducing a flankingamino acid residue at a C+1 position of an epitope to be included withinthe multi-epitope construct; (iii) introducing one or more amino acidspacer residues between two epitopes of the multi-epitope construct,wherein the spacer prevents the occurrence of a junctional epitope; and,(iv) selecting one or more multi-epitope constructs that have a minimalnumber of junctional epitopes, a minimal number of amino acid spacerresidues, and a maximum number of flanking amino acid residues at a C+1position relative to each epitope. In some embodiments, the spacerresidues are independently selected from residues that are not known HLAClass II primary anchor residues. In particular embodiments, introducingthe spacer residues prevents the occurrence of an HTL epitope. Such aspacer often comprises at least 5 amino acid residues independentlyselected from the group consisting of G, P, and N. In some embodimentsthe spacer is GPGPG (SEQ ID NO:2).

In some embodiments, introducing the spacer residues prevents theoccurrence of a CTL epitope and further, wherein the spacer is 1, 2, 3,4, 5, 6, 7, or 8 amino acid residues independently selected from thegroup consisting of A and G. Often, the flanking residue is introducedat the C+1 position of a CTL epitope and is selected from the groupconsisting of K, R, N, G, and A. In some embodiments, the flankingresidue is adjacent to the spacer sequence. The method of the inventioncan also include substituting an N-terminal residue of an epitope thatis adjacent to a C-terminus of an adjacent epitope within themulti-epitope construct with a residue selected from the groupconsisting of K, R, N, G, and A.

In some embodiments, the method of the invention can also comprise astep of predicting a structure of the multi-epitope construct, andfurther, selecting one or more constructs that have a maximal structure,i.e., that are processed by an HLA processing pathway to produce all ofthe epitopes comprised by the construct. In some embodiments, themulti-epitope construct encodes EP-HIV-1090 as set out in FIG. 9,HIV-CPT as set out in FIG. 9, or HIV-TC as set out in FIG. 9.

In another embodiment of the invention, a system for optimizingmulti-epitope constructs include a computer system having a processor(e.g., central processing unit) and at least one memory coupled to theprocessor for storing instructions executed by the processor and data tobe manipulated (i.e., processed) by the processor. The computer systemfurther includes an input device (e.g., keyboard) coupled to theprocessor and the at least one memory for allowing a user to inputdesired parameters and information to be accessed by the processor. Theprocessor may be a single CPU or a plurality of different processingdevices/circuits integrated onto a single integrated circuit chip.Alternatively, the processor may be a collection of discrete processingdevices/circuits selectively coupled to one another via either directwire/conductor connections or via a data bus. Similarly, the at leastone memory may be one large memory device (e.g., EPROM), or a collectionof a plurality of discrete memory devices (e.g., EEPROM, EPROM, RAM,DRAM, SDRAM, Flash, etc.) selectively coupled to one another forselectively storing data and/or program information (i.e., instructionsexecuted by the processor). Those of ordinary skill in the art wouldeasily be able to implement the desired computer system architecture toperform the operations and functions disclosed herein.

In one embodiment, the computer system includes a display monitor fordisplaying information, instructions, images, graphics, etc. Thecomputer system receives user inputs via a keyboard. These user inputparameters may include, for example, the number of insertions (i.e.,flanking residues and spacer residues), the peptides to be processed,the C+1 and N−1 weighting values for each amino acid, and the motifs touse for searching for junctional epitopes. Based on these inputvalues/parameters, the computer system executes a “Junctional Analyzer”software program that automatically determines the number of junctionalepitope for each peptide pair and also calculates an “enhancement” valuefor each combination of flanking residues and spacers that may beinserted at the junction of each peptide pair. The results of thejunctional analyzer program are then used in either an exhaustive orstochastic search program which determines the “optimal” combination orlinkage of the entire set of peptides to create a multi-epitopepolypeptide, or nucleic acids, having a minimal number of junctionalepitopes and a maximum functional (e.g., immunogenicity) value.

In one embodiment, if the number of peptides to be processed by thecomputer system is less than fourteen, an exhaustive search program isexecuted by the computer system which examines all permutations of thepeptides making up the polypeptide to find the permutation with the“best” or “optimal” function value. In one embodiment, the functionvalue is calculated using the equation (Ce+Ne)/J when J is greater thanzero and 2* (Ce+Ne) when J is equal to zero, where Ce is the enhancement“weight” value of an amino acid at the C+1 position of a peptide, Ne isthe enhancement “weight” value of an amino acid at the N−1 position of apeptide, and J is the number of junctional epitopes contained in thepolypeptide encoded by multi-epitope nucleic acid sequence. Thus,maximizing this function value will identify the peptide pairs havingthe least number of junctional epitopes and the maximum enhancementweight value for flanking residues. If the number of peptides to beprocessed is fourteen or more, the computer system executes a stochasticsearch program that uses a “Monte Carlo” technique to examine manyregions of the permutation space to find the best estimate of theoptimum arrangement of peptides (e.g., having the maximum functionvalue).

In a further embodiment, the computer system allows a user to inputparameter values which format or limit the output results of theexhaustive or stochastic search program. For example, a user may inputthe maximum number of results having the same function value(“MaxDuplicateFunctionValue=X”) to limit the number of permutations thatare generated as a result of the search. Since it is possible for thesearch programs to find many arrangements that give the same functionvalue, it may be desirable to prevent the output file from being filledby a large number of equivalent solutions. Once this limit is reached nomore results are reported until a larger or “better” function value isfound. As another example, the user may input the maximum number of“hits” per probe during a stochastic search process. This parameterprevents the stochastic search program from generating too much outputon a single probe. In a preferred embodiment, the number of permutationsexamined in a single probe is limited by several factors: the amount oftime set for each probe in the input text file; the speed of thecomputer, and the values of the parameters “MaxHitsPerProbe” and“MaxDuplicateFunctionValues.” The algorithms used to generate and selectpermutations for analysis may be in accordance with well-known recursivealgorithms found in many computer science textbooks. For example, sixpermutations of three things taken three at a time would be generated inthe following sequence: ABC; ACB; BAC; BCA; CBA; CAB. As a furtherexample of an input parameter, a user may input how the stochasticsearch is performed, e.g., randomly, statistically or other methodology;the maximum time allowed for each probe (e.g., 5 minutes); and thenumber of probes to perform.

Also disclosed herein are multi-epitope constructs designed by themethods described above and hereafter. The multi-epitope constructsinclude spacer nucleic acids between a subset of the epitope nucleicacids or all of the epitope nucleic acids. One or more of the spacernucleic acids may encode amino acid sequences different from amino acidsequences encoded by other spacer nucleic acids to optimize epitopeprocessing and to minimize the presence of junctional epitopes.

The invention relates to a method and system of designing multi-epitopevaccines with optimized immunogenicity. In preferred embodiments, thevaccine comprises CTL and HTL epitopes. Vaccines in accordance with theinvention allow for significant, non-ethnically biased populationcoverage, and can preferably focus on epitopes conserved amongstdifferent viral or other antigenic isolates. Through the method andsystem disclosed herein, vaccines can be optimized with regard to themagnitude and breadth of responses, and can allow for the simplestepitope configuration. Finally, general methods are provided to evaluateimmunogenicity of a multi-epitope vaccine in humans.

The method of the invention comprises designing a multi-epitopeconstruct based on principles identified herein. In one aspect, theinvention provides for simultaneous induction of responses againstspecific CTL and HTL epitopes, using single promoter multi-epitopeconstructs. Such constructs can contain many different epitopes,preferably greater than 10, often greater than 20, 25, 30, 25, 40, 45,50, 55, 60, 65, 70, or more.

In a preferred embodiment, a computer system identifies one or moreoptimal multi-epitope constructs by performing the following functionsand/or analyses:

(i) the epitopes to be incorporated into the multi-epitope construct aresorted to provide an order that minimizes the number of junctionalepitopes formed. A more detailed discussion of this sorting procedure isprovided below with reference to FIGS. 11 and 12. Preferably, as asecondary consideration in ordering epitopes, epitopes are positionedsuch that residues at the N-terminus of an epitope that promote CTLimmunogenicity are juxtaposed to the C-terminus of another CTL epitope.

(ii) flanking residues that enhance immunogenicity may be inserted atthe flanking positions of epitopes. In particular embodiments, flankingresidues are inserted at the C+1 position of CTL epitopes.

(iii) spacer sequences may be inserted between epitopes to preventoccurance of junctional epitopes. In particular embodiments, the spacersequences can also include a residue that promotes immunogenicity at theN-terminus of the linker such that the residue flanks the C-terminus ofa CTL epitope.

In particular embodiments to prevent HTL junctional epitopes, a spacercomposed of amino acid residues that do not correspond to any known HLAClass II anchor residue, are used, e.g, alternating G and P residues (aGP spacer) is included between two HTL epitopes.

Another aspect of the invention, (consideration (ii) above) involves theintroduction or substitution of particular amino acid residues atpositions that flank epitopes, e.g., a position immediately adjacent tothe C-terminus of the epitope, thereby generating multi-epitopeconstructs with enhanced antigenicity and immunogenicity compared toconstructs that do not contain the particular residue introduced orsubstituted at that site, i.e., non-optimized multi-epitope constructs.The methods of optimizing multi-epitope constructs comprise a step ofintroducing a flanking residue, preferably K, N, G, R, or A at the C+1position of the epitope, i.e., the position immediately adjacent to theC-terminus of the epitope. In an alternative embodiment, residues thatcontribute to decreased immunogenicity, i.e., negatively chargedresidues, e.g., D, aliphatic residues (I, L, M, V) or aromaticnon-trytophan residues, are replaced. The flanking residue can beintroduced by positioning appropriate epitopes to provide the favorableflanking residue, or by inserting a specific residue.

As noted in the background section, multi-epitope constructs (minigenes)encoding up to 10 epitopes have been used to induce responses against anumber of different epitopes. The data relating to an experimentalmulti-epitope construct, pMin.1 has been published (Ishioka et al., JImmunol, Vol. 162(7):3915-25 (1999)). Disclosed herein, are parametersfor designing and evaluating multi-epitope constructs with optimizedimmunogenicity that address myriad disease indications of interest.

Design parameters were identified based on a number of studies. In apreliminary evaluation of multi-epitope constructs, data on threedifferent multi-epitope constructs, incorporating 20 to 25 different CTLepitopes each, are presented (FIG. 1). One construct is based onHIV-derived epitopes, (HIV-1), while the other two incorporateHCV-derived epitopes (HCV1 and HCV2, respectively). The immunogenicityof these different multi-epitope constructs has been measured in eitherA2 or A11 HLA transgenic mice (A1, A24 and B7 restricted epitopes werenot evaluated).

Thus, eleven days after a single i.m. DNA vaccine injection, responsesagainst 8 to 14 different representative epitopes were evaluatedfollowing a single six day in vitro restimulation, utilizing assays tomeasure CTL activity (either chromium release or in situ IFN production,as described herein). Priming of epitope specific CTL could bedemonstrated for 6/8 (75%), 10/14 (72%) and 13/14 (93%) of the epitopestested in the case of HIV-1, HCV1 and HCV2, respectively. Thus,multi-epitope constructs, capable of simultaneously priming CTLresponses against a large number of epitopes, can be readily designed.However, it should be emphasized that CTL priming for some epitopes wasnot detected and, in several of the 36 cases considered, responses wereinfrequent, or varied significantly in magnitude over at least threeorders of magnitude (1000-fold). These results strongly suggested that amore careful analysis and optimization of the multi-epitope constructswas required.

The possibility that the suboptimal performance of priming for certainepitopes might be related to multi-epitope construct size was alsoexamined. In fact, most of the published reports describe multi-epitopeconstruct of up to ten epitopes, and in the few instances in which20-epitope constructs have been reported, activity directed against onlytwo or three epitopes was measured. To address this possibility, twosmaller epigene constructs (HIV-1.1 and HIV-1.2) each encompassing tenepitopes, and corresponding to one half of the HIV-1 epigene construct,were synthesized and tested. Responses against four representativeepitopes were measured.

TABLE 1 Immunogenicity appears to be independent of epigene constructsize. CTL response to different epigene constructs HIV 1 HIV 1.1 HIV 1.2(20 mer) (10 mer) (10 mer) CTL Epitope Frequency¹⁾ Magnitude²⁾ FrequencyMagnitude Frequency Magnitude Pol 774 0/8 * 0/4 * NA³⁾ NA Pol 498 18/1946.7 4/4 16.4 NA NA Gag 271  4/13  4.0 NA NA 0/4 * Env 134 5/8 16.1 NANA 4/4 14.8 ¹⁾Represents the fraction of independent cultures yieldingpositive responses ²⁾Lytic Units (LU) ³⁾Not Applicable

It was found that the responses induced by the smaller epigeneconstructs were comparable, and if anything, lower than those induced bythe twenty-epitope construct (Table 1). Accordingly, factors relating toepigene construct size are unlikely explanations for the observedsuboptimal priming to certain epitopes and thus other parameters,disclosed herein, are used to design efficacious multi-epitopeconstructs.

The Minimization of Junctional Motifs

One of the considerations in designing multi-epitope constructs is theinadvertent creation of junctional epitopes when placing epitopesadjacent to each other. The presence of such epitopes in a multi-epitopeconstruct could significantly affect performance. Strategies to guardagainst this undesired effect are disclosed herein for application tothe development of multi-epitope vaccines. Junctional epitopes can firstbe minimized by sorting the epitopes to identify an order in which thenumbers of junctional epitopes is minimized. Such a sorting procedurecan be performed using a computer or by eye, if necessary, or dependingon the number of epitopes to be included in the multi-epitope construct.

For example, a computer program that finds patterns, e.g., Panorama,manufactured by ProVUE Development, Huntington Beach, Calif., U.S.A.,can be used in accordance with one embodiment of the invention. A verylarge number of different epitope arrangements can be considered indesigning a particular multi-epitope construct. A computer programaccepts as input, the particular set of epitopes considered, and themotifs to be scanned in order to evaluate whether there are anyjunctional epitopes bearing these motifs. For example, a program cansimulate building a multi-epitope construct, and in an heuristiccomputational algorithm, examine epitope pairs to avoid or minimize theoccurrance of junctional motifs. The program can for example, evaluate6×10⁵ (about half a million) multi-epitope constructconfigurations/second.

A complete analysis of a 10-epitope construct using a computer programas described in the preceding paragraph requires examining 10factorial≅3.6×10⁶ combinations and can be completed in six seconds. Afourteen-epitope construct can be completely analyzed in a couple ofdays. Thus, analysis time goes up very rapidly as larger constructs areconsidered. However, a complete analysis is not always required and theprogram can be run for any desired length of time. In either case, thecomputer system of the present invention identifies and provides atleast one configuration having a minimum number of junctional epitopes.

An example of the results of this type of approach is presented in Table2. The number of junctional motifs in ten different random assortmentsof the same epitopes contained in the HCV1 epigene, which incorporates25 epitopes, and is the result of a two-day computer analysis, ispresented in this Table. In the non-optimized assortments, a largenumber of HLA-A2, A11 and K^(b) motifs were found, approximately 25 to38, with an average of 31. By comparison, only two such junctionalmotifs are present in the HCV1 epigene construct assortment. Inconclusion, a computer program can be utilized to effectively minimizethe number of junctional motifs present in multi-epitope constructs.

TABLE 2 Occurrence of junctional epitopes. epigene construct selectioncriteria junctional motifs HCV.a random 33 HCV.b random 26 HCV.c random28 HCV.d random 27 HCV.e random 30 HCV.f random 26 HCV.g random 38 HCV.hrandom 33 HCV.i random 33 HCV.j random 34 HCV.1 minimized 2Eliminating Class II Junctional Epitopes and Testing for Class IIRestricted Responses In Vivo

As a further element in eliminating junctional epitopes, spacersequences can be inserted between two epitopes that create a junctionalepitope when juxtaposed.

In one embodiment, to correct the problem of junctional epitopes for HTLepitopes, a spacer of, for example, five amino acids in length isinserted between the two epitopes. The amino acid residues incorporatedinto such a spacer are preferably those amino acid residues that are notknown to be primary anchor residues for any of the HLA Class II bindingmotifs. Such residues include G, P, and N. In a preferred embodiment, aspacer with the sequence GPGPG (SEQ ID NO:2) is inserted between twoepitopes. Previous work has demonstrated that the GP spacer isparticularly effective in disrupting Class II binding interactions(Sette et al., J. Immunol., 143:1268-73 (1989)). All known human ClassII binding motifs and the mouse IA^(b) (the Class II expressed by HLAtransgenic mice) do not tolerate either G or P at the main anchorpositions, which are spaced four residues apart. This approach virtuallyguarantees that no Class II restricted epitopes can be formed asjunctional epitopes.

In an example validating this design consideration, we synthesizedpolypeptides incorporating HIV-derived HTL epitopes. These epitopes arebroadly cross-reactive HLA DR binding epitopes. It was then determinedthat these epitopes also efficiently bind the murine IA^(b) Class IImolecule. A diagram illustrating the two different syntheticpolypeptides considered is shown in FIG. 2 a.

The first construct incorporates four different epitopes linearlyarranged, while the second construct incorporates the GPGPG (SEQ IDNO:2) spacer. Synthetic peptides corresponding to the three potentialjunctional epitopes were also synthesized.

The capacity of 2 nanomoles of these different constructs to prime forproliferative responses to the various epitopes in IA^(b) positive micewas tested, and compared to the responses induced by equimolar amountsof a pool of the same peptides (3 micrograms of each peptide).Specifically, groups of 3 mice were injected with peptides in CFAemulsions, 11 days after injection their lymph node cells were culturedin vitro for an additional 3 days, and thymidine incorporation wasmeasured in the last 24 hours of culture. It was found (FIG. 2 b) that,as predicted on the basis of their high affinity IA^(b) bindingcapacity, all four epitopes induced good proliferation responses.Stimulation index (SI) values in the range of 4.9 to 17.9 were observedwhen these peptides were injected in a pool. However, when the linearpolypeptide incorporating the same epitopes was tested, the responsedirected against Pol 335 was lost. This was associated with appearanceof a response directed against a junctional epitope spanning Gag 171 andPol 335. The use of the GPGPG (SEQ ID NO:2) spacer eliminated thisproblem, presumably by destroying the junctional epitope, and the Pol335 response was regained. The responses observed were of magnitudesimilar to those observed with the pool of isolated peptides.

These results demonstrate that responses against multiple HIV-derivedClass II epitopes can be simultaneously induced, and also illustrate howIA^(b)/DR crossreactivity can be utilized to investigate theimmunogenicity of various constructs incorporating HTL epitopes.Finally, they demonstrate that appropriate spacers can be employed toeffectively disrupt Class II junctional epitopes that would otherwiseinterfere with effective vaccine immunogenicity.

In the case of Class I restricted responses, one case of a naturallyoccurring junctional epitope and the consequent inhibition of epitopespecific responses has been presented by McMichael and coworkers (Tusseyet al., Immunity, Vol. 3(1):65-77 (1995)). To address the problem ofjunctional epitopes for Class I, similar analyses can be performed. Forexample, a specific computer program is employed to identify potentialClass I restricted junctional epitopes, by screening for selected murinemotifs and for the most common human Class I HLA A and B motifs.

Spacer sequences can also similarly be employed to prevent CTLjunctional epitopes. Often, very small residues such as A or G arepreferred spacer residues. G also occurs relatively infrequently as apreferred primary anchor residue (see, e.g., PCT/US00/24802) of an HLAClass I binding motif. These spacers can vary in length, e.g., spacersequences can typically be 1, 2, 3, 4, 5, 6, 7, or 8 amino acid residuesin length and are sometimes longer. Smaller lengths are often preferredbecause of physical constraints in producing the multi-epitopeconstruct.

The Influence of Flanking Regions on CTL Multi-Epitope ConstructImmunogenicity

Another factor to be considered in designing multi-epitope constructs isto insert residues that favor immunogenicity at the position flankingthe C-terminus of a CTL epitope.

Disclosed herein are studies that identify residues that increaseimmunogenicity and, accordingly, residues that are inserted inmulti-epitope constructs to optimize immunogenicity.

The molecular context in which an epitope was expressed oftendramatically influenced the frequency and/or magnitude of priming of CTLspecific for that epitope in HLA transgenic mice. Two examples are shownin Table 3.

TABLE 3 Differences in effectiveness of T cell priming for specificepitopes in different epigene constructs. Flanking Flanking SEQ SequenceSequence Immune Immune Epitope Epigene ID (N Epitope (C- ResponseResponse Identity Construct NO: terminus) Sequence terminus) FrequencyMagnitude¹⁾ Core 18 HBV.1 3 TLKAAA FLPSDFFPSV FLLSLG 6/6 5.5 pMin1 4TLKAAA FLPSDFFPSV KLTPLC 6/6 1074.5 Core 132 HCV1 5 ILGGWV DLMGYIPLVYLVAYQ  2/12 107.7 HCV2 6 VPGSRG DLMGYIPLV AKFVA 17/18 929.2¹⁾IFNγ secretory units

The immunogenicity of the HBV Core 18 epitope expressed in the pMin5epigene construct was approximately 200-fold lower in magnitude thanthat observed in the case of the pMin1 epigene construct. Similarly, theimmunogenicity of the HCV Core 132 epitope expressed in the context ofthe HCV1 epigene construct was marginal, with significant T cell primingdemonstrable in only 2 of 12 different independent CTLexperiments/cultures performed. These two positive experiments yieldedresponses of approximately 100SU of IFNγ. However, when the same epitopewas expressed in the context of the HCV2 epigene construct, positiveresponses were observed in 17/18 cases, and with average magnitudesapproximately five-fold higher.

Immunogenicity of HIV-FT in HLA-A*0201/Kb Transgenic Mice

An HIV multi-epitope DNA vaccine, HIV-FT (FIG. 3 a) encodes 20HIV-derived CTL epitopes. Of these 20 epitopes, eight are restricted byHLA-A*0201, nine by HLA-A*1101 and three by HLA-B*0702. All epitopesbound their relevant restriction element with high or moderate affinity.All of the HLA-A*0201 restricted epitopes bound purified HLA-A*0201molecules with roughly similar affinities, with IC₅₀ values in the19-192 nM range (FIG. 3 a). The HLA-A*0201 epitopes chosen for inclusionin HIV-FT are recognized in HIV-1 infected individuals and were alsohighly effective in priming for recall CTL responses when emulsifiedwith IFA and utilized to prime HLA-A*0201/K^(b) transgenic mice. Theconstruct was designed to encode the epitopes sequentially without anyintervening spacer sequences between them and a consensus IgK signalsequence was fused to the 5′ end of the construct to facilitatetransport of the encoded antigen into the endoplasmic reticulum (Ishiokaet al., J. Immunol. 162:3915-3925, 1999).

The ability of HIV-FT to prime recall CTL responses in vivo wasevaluated by intramuscular immunization of HLA-A*0201/K^(b) transgenicmice. Splenocytes from animals immunized with 100 μg of HIV-FT plasmidDNA were stimulated with each of the HLA-A*0201 epitopes encoded inHIV-FT and assayed for peptide-specific CTL activity after six days ofculture. Representative CTL responses against three of the epitopes inHIV-FT are shown in FIG. 4 a. To more conveniently compile results fromdifferent experiments the percent cytotoxicity values for eachsplenocyte culture were expressed in lytic units (Vitiello, et al., J.Clin. Invest 95:341-349, 1995). Of the eight HLA-A*0201 restrictedepitopes encoded in HIV-FT, Pol 498, Env 134, Pol 448, Vpr 62, Nef 221,and Gag 271, primed for CTL responses following DNA immunization, (FIG.4 b). The magnitude of the CTL responses varied over greater than a10-fold range, from as high as nearly 50 LU against Pol 498, too aslittle as 4 LU against Nef 221 and Gag 271. Similarly, the frequency ofrecall CTL responses varied between epitopes, with the Pol 498 epitopeinducing responses in 94% of the experiments while CTL responses to Gag271 were detected in only 31% of the experiments. In conclusion, DNAimmunization with HIV-FT, which sequentially encodes the epitopeswithout any spacer amino acids, induced recall CTL responses against themajority of the epitopes analyzed. However, the magnitude and thefrequency of the responses varied greatly between epitopes.

Correlation Between Epitope Immunogenicity and Levels of HIV-FT EpitopePresentation in Transfected Cell Lines

The differential immunogenicity of the HLA-A*0201 epitopes in HIV-FT wasthen assessed. Differential MHC binding affinity could be excluded asall of the epitopes bind HLA-A*0201 with high affinity (FIG. 3 a). Inaddition, lack of a suitable repertoire of TCR specificities inHLA-A*0201/K^(b) transgenic mice could be excluded since all epitopesyielded comparable CTL responses following immunization of HLAtransgenic mice with the optimal preprocessed peptide emulsified in IFA.Variations in the relative amounts of each epitope presented for T cellrecognition may account for the differences in epitope immunogenicity.

To test this, Jurkat cells, a human T cell line, expressing theHLA-A*0201/K^(b) gene (Vitiello et al., J. Exp. Med. 173, 1007-1015,1991) were transfected with the HIV-FT expressed in an episomal vector.A human cell line was selected for use to eliminate any possibleartifacts that may be associated with potential differences in theprocessing capabilities between humans and mice. This transfected cellline matches the human MHC presentation with human antigen processingcapabilities and provides support for the subsequent development of CTLepitope-based DNA vaccines for use in humans.

Peptide-specific CTL lines detected presentation in the transfectedtargets of four of the HLA-A*0201 epitopes encoded in the HIV-FT, Pol498, Env 134, Pol 448 and Nef 221. To quantitate the level at which eachof these epitopes was produced and presented, the CTL lines specific forthe various epitopes were incubated with untransfected targets andvariable amounts of each epitope or peptides. These CTL dose responsecurves were utilized as standard curves to determine the peptideconcentration inducing levels of IFNγ secretion equivalent to thoseobserved in response to the HIV-FT transfected target cells. This valueis referred to as a “peptide equivalent dose” and taken as a relativemeasure of the amount of epitope presented on the transfected cell.

Table 4 summarizes the findings of this analysis for eight of theHLA-A*0201 epitopes encoded in the HIV-FT. Peptide equivalent dosesvaried from a high of 33.3 ng/ml for Nef 221 to less than 0.4 ng/mlpeptide equivalents for epitopes Gag 271, Gag 386 and Pol 774.Cumulatively these results indicate that in human cells linestransfected with HIV-FT there is at least a 100-fold variation exists inthe levels of presentation of the different HLA-A*0201 restrictedepitopes. All of the epitopes that were presented at detectable levelsin antigenicity assays were also immunogenic in vivo. The only epitopethat was immunogenic and not antigenic was Gag 271. In this case,immunization of HLA-A*0201/K^(b) transgenic mice with HIV-FT induced aweak CTL response in less than a third of the cultures tested. The othertwo epitopes, which were presented below the limit of sensitivity forthe antigenicity analysis, Gag 386 and Pol 774, were non-immunogenic. Inconclusion these results suggest that the heterogeneity in CTL responsesinduced by HIV-FT immunization can at least in part be attributed tosuboptimal epitope presentation.

TABLE 4 Comparison of HIV-FT immunogenicity and antigenicity HIV-FTAntigenicity HIV-FT Immunogenicity Peptide Epitope magnitude¹ frequency²Equivalents³ n⁴ Pol 498 58.8 (2.2) 94% (16/17) 23.8 (2.0) 4 Env 134 16.1(5.0) 63% (5/8)  6.2 (1.2) 3 Pol 448 15.7 (2.6) 54% (7/13) 24.7 (3.9) 3Vpr 62  9.9 (1.9) 83% (10.12) ND — Nef 221  4.4 (1.3) 78% (7/9) 33.3(6.0) 3 Gag 271  4.0 (1.4) 31% (4/13) <0.4 6 Gag 386 0  0% (0/17) <0.4 3Pol 774 0  0% (0/8) <0.4 1 ¹magnitude expressed as LU (ref); thecorrelation coefficient relative to peptide equivalents R + 0.44²frequency of positive cultures (number cultures >2LU/total tested); thecorrelation coefficient relative to peptide equivalents R + 0.8.³magnitude expressed in ng/ml ⁴number of independent experimentsFlanking Amino Acids Influence CTL Epitope Immunogenicity In VivoFollowing Vaccination

As described herein, the particular amino acids flanking individual CTLepitopes is one factor that influences or enhances the efficiency withwhich an epitope is processed by altering the susceptibility of theantigen to proteolytic cleavage. To examine the influence of flankingamino acids on epitope immunogenicity, immunogenicity data was obtainedfrom HLA-A*0201, -A*1101 and -B*0701 transgenic mice immunized with anumber of unrelated experimental multi-epitope DNA constructs encodingminimal CTL epitopes without intervening sequences. A databaserepresenting 94 different epitope/flanking residue combinations wascompiled to determine the possible influence the immediately flankingamino acids on epitope immunogenicity. A given epitope and flankingamino acid combination was included only once to prevent artificialskewing of the analysis because of redundancies. Epitope immunogenicityin HLA transgenic was considered optimal if greater than 100 SU or 20 LUin at least 30% of the cultures measured. CTL responses were typicallyscored in one of four categories: (+++), outstanding-more than 200 LU or1000 SU; (++), good-20-200 LU or 100-1000 SU; (+), intermediate-2 to 20LU or 10 to 100 SU; and (+/−), weak or negative-less than 2 LU or 10 SU.The numbers of optimal versus sub-optimal responses were categorizedbased on the chemical type of amino acid in the flanking positions andthe significance of differences were determined using a chi-square test.

This analysis did not find any associations between the type of aminoacids present at the amino-terminus of an epitope and immunogenicity.However, significant effects of the carboxyl-terminus flanking residue,the C+1 residue, were identified. Positively charged amino acids, K or Rwere most frequently associated with optimal CTL responses, a frequencyof 68% (FIG. 5). The presence of amino acids N and Q at the C+1 residuewas also associated with strong CTL responses in 55.5% of the casesexamined; when epitopes were flanked at the C+1 position by N, theyinduced optimal CTL responses in ¾ cases. In general, small residuessuch as C, G, A, T, and S promoted intermediate CTL responses inducingstrong responses in 54% of the combinations available for analysis.Conversely, epitopes flanked by aromatic and aliphatic amino acidsinduced optimal in vivo responses in only 36% and 17% of the cases,respectively. The negatively charged residue, D, yielded a suboptimalCTL response. The influence of C+1 amino acid on epitope immunogenicitywas found to be statistically significant using a chi-square test(P<0.03). No significant influence on epitope immunogenicity was notedwhen similar analysis was performed for C-terminal residues more distalthan the C+1 position.

Direct Evaluation of the Effect of the C1 Residue on EpitopeImmunogenicity

To directly evaluate the effect of preferred versus deleterious types ofamino acids in the C+1 flanking position, two multi-epitope constructs,referred to as HBV.1 and HBV.2 (FIG. 3 b) were evaluated. As withHIV-FT, these HBV constructs encode the epitopes sequentially withoutintervening spacers. The HBV.1 and HBV.2 epigenes were generated byreplacing the HIV-1 epitopes in pMin1 (an experimental multi-epitopeconstruct previously characterized (Ishioka, supra) with similarHBV-derived epitopes).

For HBV.1, the HIV-1 epitope directly following the highly immunogenicHBV Core 18 epitope was replaced with the HBV Pol 562 epitope. Thisaltered the C+1 residue from a K to an F. The second construct, HBV.2,was produced by the insertion of an additional epitope, HBV Pol 629,between the HBV Core 18 and Pol 562 epitopes; a change that replaced theC+1 amino acid with a K residue. When the immunogenicity of the Core 18epitope presented in these different contexts was evaluated inHLA-A*0201/K^(b) transgenic mice, it was determined that the Core 18epitope was virtually non-immunogenic in HBV.1 but strongly immunogenicin HBV.2 (FIG. 6 a). The reduction of in vivo immunogenicity for thisepitope was as would be predicted by our previous analysis.

To further test the effects of the C+1 flanking amino acid on CTLepitope immunogenicity, a set of constructs that differ from HBV.1 bythe insertion of single amino acids at the C+1 position relative to theCore 18 epitope (FIG. 3 b) was evaluated. Little or no CTL response wasobserved against the Core 18 epitope when flanked at the C+1 position byW, Y, or L (FIG. 6 b). In contrast, insertion of a single K residuedramatically increased the CTL response to Core 18. The responses werecomparable to those observed in HBV.2, where the Core 18 epitope isflanked by Pol 629 epitope (Pol 629 has a K at the N-terminus).Enhancement of the Core 18 CTL response was also observed to insertionof R, C, N, or G. The effect of these insertions is specific, as theimmunogenicity of other epitopes within these constructs did not exhibitsignificant changes in CTL responses (data not shown). In conclusion,these data indicate that the C+1 amino acid can dramatically influenceepitope immunogenicity.

Variations in CTL Epitope Immunogenicity are Correlated with the AmountPresented

If the variation of the immunogenicity of Core 18 associated withdifferent C+1 residues was the result of differential sensitivity toproteolytic cleavage then large differences in the levels of epitopepresentation should be detectable in different constructs. To test this,Jurkat cells, expressing the same HLA-A*0201/K^(b) gene expressed in thetransgenic mice, were transfected with an episomal vector expressingeither HBV.1 or HBV.1K. The Core 18 epitope was presented at >10⁵ higherlevels when a K was in the C+1 position, compared to the presence of anF in the same position (FIG. 7). It is unlikely that this difference inCore 18 presentation is attributed to differences in gene expressionbetween target cell lines since presentation of Pol 455 varied by lessthan ten-fold. These data demonstrate the striking effect that aminoacids at the C+1 position can exert on efficiency of epitopepresentation in multi-epitope DNA vaccines. Thus, these data show thatthe immunogenicity of CTL epitopes in a DNA vaccine can be optimizedthrough design considerations that affect the level of epitopepresentation. This type of optimization is applicable to epitope-basedvaccines delivered using other formats, such as viral vectors as well asother expression vectors known to those of skill in the art, since theeffects are exerted after the antigen is transcribed and translated.

In summary, for flanking residues, it was found that either very smallresidues such as A, C or G, or large residues such as Q, W, K, or R weregenerally associated with good or outstanding responses. The absence ofa C+1 residue because of a stop codon in the multi-epitope construct, orthe presence of intermediate size residues such as S or T was associatedwith a more intermediate response pattern. Finally, in the case of anegatively charged residue, D; aliphatic (V, I, L, M) or aromaticnon-tryptophan residues (Y, F), relatively poor responses were observed.These results show that the particular residue flanking the epitope'sC-terminus can dramatically influence the response frequency andmagnitude. Flanking residues at the C+1 position can also be introducedin combination with spacer sequences. Thus, a residue that favorsimmunogenicity, preferably, K, R, N, A, or G, is included as a flankingresidue of a spacer.

Sorting and Optimization of Multi-Epitope Constructs

To develop multi-epitope constructs using the invention, the epitopesfor inclusion in the multi-epitope construct are sorted and optimizedusing the parameters defined herein. Sorting and optimization can beperformed using a computer or, for fewer numbers of epitopes, not usinga computer.

Computerized optimization can typically be performed as follows. Thefollowing provides an example of a computerized system that identifiesand optimizes, e.g., provides for a minimal number of junctionalepitopes and a maximal number of flanking residues, epitopecombinations. FIG. 10 illustrates a computer system 100 for performingthe optimization of multi-epitope constructs, in accordance with oneembodiment of the invention. The computer system 100 may be aconventional-type computer which includes processing circuitry, e.g., acentral processing unit (CPU), memory, e.g., a hard disk drive (ROM), arandom access memory (RAM), cache, and other components, devices andcircuitry (not shown) typically found in computers today. In a preferredembodiment, the computer system 100 includes, among other components anddevices, a Macintosh G3 333 MHz processor, a six Gigabyte (GB) harddrive, 96 Megabytes of RAM, and 512 Kilabytes (KB) of cache memory,capable of searching 600,000 to 700,000 permutations per second. Thecomputer system 100 further includes a monitor 102 for displaying text,graphics and other information to a user and a keyboard 104 for allowinga user to input data, commands, and other information to the computersystem 100.

As shown in FIG. 10, in one embodiment, the computer system 100 maycommunicate with one or more remote computers 150 through a computernetwork 160 such that registered users at remote locations can performthe junctional analyses and multi-epitope construct optimizationprocedures described herein by logging on at the remote computers 150and supplying a required password or user identification. The computernetwork 160 may be a local area network (LAN), a wide area network(WAN), or the world-wide web (i.e., Internet). These types of networksare well-known in the art and, therefore, a discussion of these networksand their communication protocols is not provided herein.

In a preferred embodiment, the computer system 100 stores a softwareprogram, e.g., object code, in the hard drive memory of the computersystem 100. This object code is executed by the CPU for performing thefunctions described herein. One component, or module, of the softwareprogram carries out the function of analyzing and identifying junctionalepitopes at the peptide junctions of the polypeptide encoded by amulti-epitope nucleic acid construct as well as evaluating combinationsof spacer and flanking residues at these junctions. This software moduleis referred to herein as the “Junctional Analyzer” module or program. Ina preferred embodiment, the Junctional Analyzer analyzes and processespeptides entered by a user in accordance with other criteria, data andoperating parameters described below.

FIGS. 11A-B (hereinafter FIG. 11) illustrate an exemplary input textfile 200 containing user input data and parameters which is used by theJunctional Analyzer program, in accordance with one embodiment of theinvention. As shown in FIG. 11, various types of input data are providedto the program. First, a user may enter a set of peptides or epitopes202 for processing. A set of weights 204 for each amino acid, when itappears in a C+1 and N−1 position, is also entered into the text file bythe user. In one embodiment, the weight values are determined bystatistical or empirical analysis of experimental results reflecting theimmunogenicity or antigenicity “enhancement” effects of each amino acidwhen it is placed at the C+1 or N−1 positions of a polypeptide. However,the assignment of weight values for each amino acid may be performed byany number of methodologies, including in vitro and in vivo studies,which would be apparent to those of ordinary skill in the art, dependingon the desired criteria used to determine the weight values. Someexamples of such experiments or studies are described in further detailbelow.

In a preferred embodiment, a database containing differentepitope/flanking residue combinations is stratified on the basis ofepitope immunogenicity and the number of optimal versus suboptimalresponses is sorted to rank the amino acids and assign enhancementweight values. The text file also contains a set of motifs 206 to use indetecting junctional epitopes. In a preferred embodiment, the user mayalso enter a maximum number of amino acids (spacers and flanking) toinsert between each pair of peptides (MaxInsertions) 208 to function asspacers and/or flanking residues. Other parameters, values or commands(collectively referred to herein as “parameters”) to control theoperation of the program may also be entered such as, for example:“OutputToScreen (Y/N)” 210; “OutputToFile (Y/N)” 212; the minimumfunction value to accept as a valid result (“MinimumAccepted”) 214; themaximum number of results having the same function value(“MaxDuplicateFunctionValue”) 216; the maximum time allowed for a searchin minutes (“SearchTime”) 218; whether an Exhaustive Search is desired(“Exhaustive ═Y/N”) 220; the number of Stochastic search probes(“NumStochasticProbes”) 222; the maximum number of hits allowed persingle probe during a stochastic search (“MaxHitsPerProbe”) 224; andwhether the start of each probe should be random or other(“RandomProbeStart(Y/N)”) 226. These parameters are provided forpurposes of illustration only. Other parameters to control the operationand output format of the program may be entered as would be obvious tothose of ordinary skill in the art.

The motifs 206 in the text file 200 provide a “mask” or structural modelfor identifying junctional epitopes. For example the first motif 206 ashown in FIG. 11, XXXX(F or Y)XX(L, I, M or V), defines an epitope thatis eight amino acids in length. The value “X” indicates that any aminoacid may be at that position of the epitope. The value “(F or Y)”indicates that either an F amino acid or a Y amino acid may be in thefifth position of the epitope. Similarly, “(L, I, M or V)” indicatesthat any one of the listed amino acids, L, I, M or V, may be in theeighth position of the epitope. Therefore if a sequence of eight aminoacids spanning a junction of two peptides satisfies the above motifcriteria, it is identified as a junctional epitope.

FIG. 12 illustrates a flow chart diagram of one embodiment of theJunctional Analyzer program. At step 301, the program receives userinputs and instructions for performing the junctional analysisoperation. In a preferred embodiment, the program uses an input textfile 200 as shown in FIG. 11 to input parameters 202-226. As iswell-known in the art, such a text file may be derived, for example,from a Microsoft Excel™ spreadsheet file or document, to specify desiredinput parameters (e.g., epitopes, motifs, flanking residue weightvalues, maximum number of hits, maximum search time, etc.) for itsoperation. At step 303, the Junctional Analyzer program generates a listof all epitope pairs. For example, if ten epitope sequences are enteredby the user, there will be a total of ninety (10×9) epitope (peptide)pairs. Next, at step 305, for each pair of peptides or epitopes, theprogram determines the set of insertions that results in the minimumnumber of junctional epitopes and/or the maximum effect from the C+1 andN−1 contribution of spacing residues. To make this determination, theprogram calculates a function value for each possible combination ofspacers for each peptide pair, where the number of spacers can rangefrom 0 to MaxInsertions 208 (FIG. 11) and any arrangement of known orprespecified amino acids may be considered. In a preferred embodiment,the function value is calculated using the following equation:F=(C+N)/J, where C is the enhancement weight value for a flanking aminoacid located at the C+1 position of an epitope, N is the enhancementweight value for a flanking amino acid located at the N−1 position of anepitope, and J is the number of junctional epitopes present. Sincemultiple motifs may be satisfied at one junction of a peptide pair, Jmay be a number greater than one. When J=0, F=2(C+N). This secondequation was chosen because for a fixed value of (C+N), the functionvalue F will double when J changes from two to one, and will doubleagain when J changes from one to zero. It is understood, however, thatthe above equations are exemplary only and that other equations forevaluating peptide pairs can be easily added to the program at any time.Modifications or changes to the above equations, depending on thedesired criteria for emphasis or evaluation, would be readily apparentto those of ordinary skill in the art. At step 307, the program outputsthe optimum combination of insertions (spacing and/or flanking residues)for each pair of peptides and the maximum function value for each pairof peptides. In a preferred embodiment, at step 307, the output fromthis program is generated as an output text file that lists, for eachpair of peptides, the insertion that yields the maximum function result.

FIGS. 13A-D (hereinafter FIG. 13) illustrate an exemplary output textfile 400 that lists, for each peptide pair, the spacer combinationhaving the maximum function value. In the example shown in FIG. 13,eleven peptides, labeled A-K 202 (FIG. 11), were processed, the Motifs206 were used to detect junctional epitopes, the enhancement weightvalues for each potential flanking residue 204 were used, andMaxInsertions 208 was set to four. Other parameters for controlling theoperation and format of the Junctional Analyzer program were set asillustrated by the parameter settings 402. For purposes of convenience,in a preferred embodiment, these input parameters are repeated in theoutput text file 400. The output text file 400 includes an output table404 which contain the results of steps 305 (FIG. 12). The first column(Col. 1) of the output table 404 indicates the first peptide of a pair.The second column (Col. 2) of the output table lists the first aminoacid insertions that function both as a spacer and the C+1 flankingamino acid. The third column lists a second spacer amino acid. Thefourth column lists a third spacer amino acid. The fifth column lists afourth spacer amino acid that is also the N−1 flanking amino acid forthe second peptide of the pair which is listed in column six. Theseventh column lists the enhancement weight value of the C+1 flankingamino acid listed in column two. The eighth column lists the enhancementweight value of the N−1 flanking amino acid listed in column six 412.The ninth column lists the sum of the C+1 and N−1 enhancement weightvalues. The tenth column lists the number of junctional epitopes foundin the peptide pair and the eleventh column lists the maximum functionvalue for the peptide pair based on the equations listed above. Forexample, the first row of the output table 404 shows that for thepeptide pair A-B, corresponding to the peptides VLAEAMSQV (SEQ IDNO:7)-ILKEPVHGV (SEQ ID NO:8), the spacer combination of three aminoacids, CAL, eliminates all junctional epitopes and provides a maximumfunction value of 8.80. It is understood, however, that other outputoptions may be implemented in accordance with the invention. Forexample, the output table 404 may show the top 32 results for each pairof peptides, or show every result for all possible insertions in theorder evaluated, or trace the motif search process to generate largeoutput files, depending on the level of detail and/or analysis desiredby the user.

In a preferred embodiment, the information contained in the output table404 is used to perform either an “Exhaustive J Search” or a “StochasticJ Search” to identify a polypeptide construct linking all elevenpeptides, including optimum spacer combinations. For eleven peptides,for example, there will be ten junctions. Therefore, the permutationthat yields the largest sum of function values taking into account allten junctions is identified as the “optimum” permutation(s) of themulti-epitope constructs. In one embodiment, for the convenience of theuser, the output text file 400 will also contain the original list ofpeptides/epitopes 202, the weight values used 204, the motifs used 206,and MaxInsertion value 208, and other parameter settings 402 enteredinto the input text file 200 of FIG. 11.

The “Exhaustive J Search” looks at all permutations of the peptides andselects the ones that have the largest function sum. However, due to thefactorial nature of permutations, as the number of peptides to beprocessed increases, the time required to complete an “Exhaustive JSearch” increases almost exponentially. For example, using a standardMacintosh 333 MHz computer, the estimated running time for 13 peptidesis approximately 2.9 hours and would be approximately 40 hours for 14peptides. The “Stochastic J Search” is designed to search many areas ofthe permutation sequence, rather than the entire permutation space, andreport the best function sum that it finds. By reporting onlypermutations that meet or exceed the current maximum function total, itis possible to search a much broader area of the permutation sequence.This technique has been successful with as many as 20 peptides. The timeto perform an exhaustive search of 20 peptides is estimated to be on theorder of 1.3×10⁵ years.

Referring again to FIG. 12, at step 309, the program determines whetherto perform an “Exhaustive J Search” or a “Stochastic J Search” of thepossible permutations of polypeptides from the output text file 400. Ina preferred embodiment, the determination at step 309 is made by theuser who inputs whether the search will be Exhaustive or Stochastic asindicated by the input parameter, Exhaustive (Y/N) 220 (FIG. 11). Inother embodiments, the program may automatically select either aStochastic or Exhaustive search depending on the number of peptides tobe processed. For example, if less than 14 epitopes are to be included,an “Exhaustive J Search” routine is automatically selected by theprogram. The Exhaustive search program examines all permutations of theepitopes making up the multi-epitope construct to find the one(s) withthe best value for the sum of the optimizing function for all pairs ofepitopes. This is guaranteed to find the “best” permutation(s) since allare examined. If 14 or more epitopes are to be included in themulti-epitope construct, a “Stochastic J Search” is used. In a preferredembodiment, the “Stochastic J Search” uses a Monte Carlo technique,known to those of skill in the art, to examine many regions of thepermutation space to find the best estimate of the optimum arrangementof the peptides. However, other methods of Stochastic searching may beimplemented in accordance with the invention. For example, rather thanrandomly picking a starting permutation for each stochastic probe, theprogram may require that each probe begin with a permutation beginningwith a different one of the peptides entered by the user. For example,if there were just three peptides, A, B and C, the three probes wouldbegin with, for example, ABC, BAC and CBA. This method provides a fairlyuniform coverage of the possible permutations.

If a “Stochastic J Search” has been selected, next, at step 311, theprogram begins the Stochastic search by initiating a probe. Next, atstep 313, the program determines if the maximum search time per probehas been exceeded. If the maximum search time has not been reached,next, at step 315, the program determines whether a single probe hasreached or exceeded the maximum number of “hits” per probe. In oneembodiment, a probe hit is registered when a permutation's functionvalue sum is the same as or greater than the largest function sumpreviously registered for one or more previously analyzed permutations.If the maximum number of hits per probe has not been reached, then, atstep 317, the current stochastic probe evaluates the next permutation orset of permutations and the process returns step 313. If at step 315 itis determined that the maximum number of hits per probe has been reachedor exceeded, then, the program proceeds to step 319, where the programdetermines whether a maximum number of probes have already beenexecuted. Also, if at step 313, it is determined that the maximum timelimit per probe has been reached or exceeded, the program proceeds tostep 319 to determine if the maximum number of probes have beencompleted. If, at step 319, it is determined that the maximum number ofprobes has not been reached, the program returns to step 311 and a newsearch probe is initiated. If at step 319 it is determined that themaximum number of probes have been executed, the program then proceedsto step 323 where it outputs the best set of optimum permutationsidentified up to that point. This “best set” may consist of only thosepermutations having the highest function sum or, alternatively, mayconsist of the permutations having the top three highest function sums,for example, or any other output criteria desired by the user.

In one preferred embodiment, if a probe has received a maximum number ofhits specified per probe, any unused time for that probe is divided bythe remaining probes to decide how much time should be allocated to eachof the remaining probes. In other words, if a probe terminates earlybecause of finding too many hits then the remaining probes are allocatedmore time. This functionality can be easily implemented by those ofordinary skill in the computer programming arts.

If at step 309, an Exhaustive search has been selected, then, at step321, an exhaustive search is initiated which analyzes every permutation,as described above. At the completion of the Exhaustive analysis, theprogram proceeds to step 323 where it outputs the “best set” of optimumpermutations found. As mentioned above, this “best set” may includethose permutations with the highest sum function values, or the topthree highest sum function values, or permutations meeting any desiredcriteria specified by the user (e.g., top 30 permutations with thehighest function values).

For each of the decision steps or determination steps discussed above(e.g., steps 313, 315 and 319), the program may be set to perform aquery at periodic intervals (e.g., every five seconds) or,alternatively, the program may be set to perform a query after aspecified number of permutations (e.g., five) have been analyzed orafter every permutation has been analyzed. Any one of these operationand timing protocols is easily implemented and adjusted by those ofordinary skill in the art.

The Program output provides a list of the best arrangements of theepitopes. Since many permutations may have the same value of theevaluation function, several are generated so that other factors can beconsidered in choosing the optimum arrangement. Examples ofmulti-epitope constructs generated using the above-describedcomputerized techniques are illustrated in FIG. 9. An exemplary processflow implemented by the method and system of the invention is providedabove. As would be readily apparent to those of ordinary skill, otherfactors such as charge distribution, hydrophobic/hydrophilic regionanalysis, or folding prediction could also be incorporated into theevaluation function to further optimize the multi-epitope constructs. Inaddition, the multi-epitope construct may be further optimized byprocessing a multi-epitope construct already optimized by the processthrough the same or similar process one or more additional times. In thesubsequent rounds of processing one or more parameters may be modifiedas compared to the parameters used in the first round of optimization.An example of a multi-epitope construct that was optimized in two roundsis the HBV-30CL construct.

Multi-epitope constructs can also be optimized by considering theresulting macromolecular structure. Macromolecular structures such aspolypeptide structures can be described in terms of various levels oforganization. For a general discussion of this organization, see, e.g.,Alberts et al., Molecular Biology of the Cell (3^(rd) ed., 1994) andCantor and Schimmel, Biophysical Chemistry Part I. The Conformation ofBiological Macromolecules (1980). “Primary structure” refers to theamino acid sequence of a particular peptide. “Secondary structure”refers to locally ordered, three dimensional structures, within apolypeptide. These structures are commonly known as domains. Domains areportions of a polypeptide that form a compact functional unit of thepolypeptide. Typical domains are formed by combinations of secondarystructure (e.g., β-sheets and α-helices). “Tertiary structure” refers tothe complete three-dimensional structure of a polypeptide monomer.“Quaternary structure” refers to the three dimensional structure formedby the noncovalent association of independent tertiary units.

Structural predictions such as charge distribution,hydrophobic/hydrophilic region analysis, or folding predictions can beperformed using sequence analysis programs known to those of skill inthe art, for example, hydrophobic and hydrophilic domains can beidentified (see, e.g., Kyte & Doolittle, J. Mol. Biol. 157:105-132(1982) and Stryer, Biochemistry (3^(rd) ed. 1988); see also any of anumber of Internet based sequence analysis programs, such as those foundat dot.imgen.bcm.tmc.edu.

A three-dimensional structural model of a multi-epitope construct canalso be generated. This is generally performed by entering amino acidsequence to be analyzed into a predictive computer system that cangenerate a model. The amino acid sequence represents the primarysequence or subsequence of the protein, which encodes the structuralinformation of the protein. The three-dimensional structural model ofthe protein is then generated by the interaction of the computer system,using software known to those of skill in the art.

The amino acid sequence represents a primary structure that encodes theinformation necessary to form the secondary, tertiary and quaternarystructure of the protein of interest. The software looks at certainparameters encoded by the primary sequence to generate the structuralmodel. These parameters are referred to as “energy terms,” and primarilyinclude electrostatic potentials, hydrophobic potentials, solventaccessible surfaces, and hydrogen bonding. Secondary energy termsinclude van der Waals potentials. Biological molecules form thestructures that minimize the energy terms in a cumulative fashion. Thecomputer program is therefore using these terms encoded by the primarystructure or amino acid sequence to create the secondary structuralmodel. The tertiary structure of the protein encoded by the secondarystructure is then formed on the basis of the energy terms of thesecondary structure. The user can enter additional variables such aswhether the protein is membrane bound or soluble, its location in thebody, and its cellular location, e.g., cytoplasmic, surface, or nuclear.These variables along with the energy terms of the secondary structureare used to form the model of the tertiary structure. In modeling thetertiary structure, the computer program matches hydrophobic faces ofsecondary structure with like, and hydrophilic faces of secondarystructure with like. Those multi-epitope constructs that are mostreadily accessible to the HLA processing apparatus are then selected.

Assessment of Immunogenicity of Multi-Epitope Vaccines

The development of multi-epitope constructs represents a uniquechallenge, because the species-specificity of the peptide binding toMHC. Different MHC types from different species tend to bind differentsets of peptides (Rammensee et al., Immunogenetics, Vol. 41(4):178-228(1995)). As a result, it is not possible to test in regular laboratoryanimals a construct composed of human epitopes. Alternatives to overcomethis limitation are generally available. They include: 1) testinganalogous constructs incorporating epitopes restricted by non-human MHC;2) reliance on control epitopes restricted by non human MHC; 3) relianceon crossreactivity between human and non-human MHC; 4) the use of HLAtransgenic animals; and 5) antigenicity assays utilizing human cells invivo. The following is a brief overview of the development of thetechnology for analyzing antigenicity and immunogenicity.

Class I HLA Transgenic Mice

The utility of HLA transgenic mice for the purpose of epitopeidentification (Sette et al., J Immunol, Vol. 153(12):5586-92 (1994);Wentworth et al., Int Immunol, Vol. 8(5):651-9 (1996); Engelhard et al.,J Immunol, Vol. 146(4):1226-32 (1991); Man et al., Int Immunol, Vol.7(4):597-605 (1995); Shirai et al., J Immunol, Vol. 154(6):2733-42(1995)), and vaccine development (Ishioka et al., J Immunol, Vol.162(7):3915-25 (1999)) has been established. Most of the publishedreports have investigated the use of HLA A2.1/K^(b) mice but it shouldbe noted that B*27, and B*3501 mice are also available. Furthermore, HLAA*11/K^(b) mice (Alexander et al., J Immunol, Vol. 159(10):4753-61(1997)), and HLA B7/K^(b) and HLA A1/K^(b) mice have also beengenerated.

Data from 38 different potential epitopes was analyzed to determine thelevel of overlap between the A2.1-restricted CTL repertoire ofA2.1/K^(b)-transgenic mice and A2.1+ humans (Wentworth et al., Eur JImmunol, Vol. 26(1):97-101 (1996)). In both humans and mice, an MHCpeptide binding affinity threshold of approximately 500 nM correlateswith the capacity of a peptide to elicit a CTL response in vivo. A highlevel of concordance between the human data in vivo and mouse data invivo was observed for 85% of the high-binding peptides, 58% of theintermediate binders, and 83% of the low/negative binders. Similarresults were also obtained with HLA A11 and HLA B7 transgenic mice(Alexander et al., J Immunol, Vol. 159(10):4753-61 (1997)). Thus,because of the extensive overlap that exists between T cell receptorrepertoires of HLA transgenic mouse and human CTLs, transgenic mice arevaluable for assessing immunogenicity of the multi-epitope constructsdescribed herein.

The different specificities of TAP transport as it relates to HLA A11mice does not prevent the use of HLA-A11 transgenic mice of evaluationof immunogenicity. While both murine and human TAP efficiently transportpeptides with an hydrophobic end, only human TAP has been reported toefficiently transport peptides with positively charged C terminal ends,such as the ones bound by A3, A11 and other members of the A3 supertype.This concern does not apply to A2, A1 or B7 because both murine andhuman TAP should be equally capable of transporting peptides bound byA2, B7 or A1. Consistent with this understanding, Vitiello (Vitiello etal., J Exp Med, Vol. 173(4):1007-15 (1991)) and Rotzschke (Rotzschke O,Falk K., Curr Opin Immunol, Vol. 6(1):45-51 (1994)) suggested thatprocessing is similar in mouse and human cells, while Cerundolo(Rotzschke O, Falk K., Curr Opin Immunol, Vol. 6(1):45-51 (1994))suggested differences in murine versus human cells, both expressing HLAA3 molecules. However, using HLA A11 transgenics, expression of HLAmolecules on T and B cells in vivo has been observed, suggesting thatthe reported unfavorable specificity of murine TAP did not preventstabilization and transport of A11/K^(b) molecules in vivo (Alexander etal., J Immunol, Vol. 159(10):4753-61 (1997)). These data are inagreement with the previous observation that peptides with a chargedC-termini could be eluted from murine cells transfected with A11molecules (Maier et al., Immunogenetics; Vol. 40(4):306-8 (1994)).Responses in HLA A11 mice to complex antigens, such as influenza, andmost importantly to A11 restricted epitopes encoded by multi-epitopeconstructs (Ishioka et al., J Immunol, Vol. 162(7):3915-25 (1999)) hasalso been detected. Thus, the TAP issue appears to be of minor concernwith transgenic mice.

Another issue of potential relevance in the use of HLA transgenic miceis the possible influence of β2 microglobulin on HLA expression andbinding specificity. It is well known that human β2 binds both human andmouse MHC with higher affinity and stability than mouse β2 microglobulin(Shields et al., Mol Immunol Vol. 35(14-15):919-28 (1998)). It is alsowell known that more stable complexes of MHC heavy chain and β2facilitate exogenous loading of MHC Class I (Vitiello et al., Science,Vol. 250(4986):1423-6 (1990)). We have examined the potential effect ofthis variable by generating mice that are double transgenics forHLA/K^(b) and human β2. Expression of human β2 was beneficial in thecase HLA B7/K^(b) mice, and was absolutely essential to achieve goodexpression levels in the case of HLA A1 transgenic mice. Accordingly,HLA/K^(b) and β2 double transgenic mice are currently and routinely bredand utilized by the present inventors. Thus, HLA transgenic mice can beused to model HLA-restricted recognition of four major HLA specificities(namely A2, A11, B7 and A1) and transgenic mice for other HLAspecificities can be developed as suitable models for evaluation ofimmunogenicity.

Antigenicity Testing for Class I Epitopes

Several independent lines of experimentation indicate that the densityof Class I/peptide complexes on the cell surface may correlate with thelevel of T cell priming. Thus, measuring the levels at which an epitopeis generated and presented on an APC's surface provides an avenue toindirectly evaluate the potency of multi-epitope nucleic acid vaccinesin human cells in vitro. As a complement to the use of HLA Class Itransgenic mice, this approach has the advantage of examining processingin human cells. (Ishioka et al., J Immunol, Vol. 162(7):3915-25 (1999))

Several possible approaches to experimentally quantitate processedpeptides are available. The amount of peptide on the cell surface can bequantitated by measuring the amount of peptide eluted from the APCsurface (Sijts et al., J Immunol, Vol. 156(2):683-92 (1996); Demotz etal., Nature, Vol. 342(6250):682-4 (1989)). Alternatively, the number ofpeptide-MHC complexes can be estimated by measuring the amount of lysisor lymphokine release induced by infected or transfected target cells,and then determining the concentration of peptide necessary to obtainequivalent levels of lysis or lymphokine release (Kageyama et al., JImmunol, Vol. 154(2):567-76 (1995)).

A similar approach has also been used to measure epitope presentation inmulti-epitope nucleic acid-transfected cell lines. Specifically,multi-epitope constructs that are immunogenic in HLA transgenic mice arealso processed into optimal epitopes by human cells transfected with thesame constructs, and the magnitude of the response observed intransgenic mice correlates with the antigenicity observed with thetransfected human target cells (Ishioka et al., J Immunol, Vol.162(7):3915-25 (1999)).

Using antigenicity assays, a number of related constructs differing inepitope order or flanking residues can be transfected into APCs, and theimpact of the aforementioned variables on epitope presentation can beevaluated. This can be a preferred system for testing where a relativelylarge number of different constructs need to be evaluated. Although itrequires large numbers of epitope-specific CTLs, protocols that allowfor the generation of highly sensitive CTL lines (Alexander-Miller etal., Proc Natl Acad Sci USA, Vol. 93(9):4102-7 (1996)) and also fortheir expansion to large numbers (Greenberg P. D., Riddell S. R.,Science, Vol. 285(5427):546-51 (1999)) have been developed to addressthis potential problem.

It should also be kept in mind that, if the cell selected for thetransfection is not reflective of the cell performing APC function invivo, misleading results could be obtained. Cells of the B cell lineage,which are known “professional” APCs, are typically employed astransfection recipients. The use of transfected B cells of this type isan accepted practice in the field. Furthermore, a good correlation hasalready been noted between in vitro data utilizing transfected human Bcells and in vivo results utilizing HLA transgenic mice, as described inmore detail herein.

Measuring HTL Responses

In preferred embodiments, vaccine constructs are optimized to induceClass II restricted immune responses. One method of evaluatingmulti-epitope constructs including Class II epitopes, is to use HLA-DRtransgenic mice. Several groups have produced and characterized HLA-DRtransgenic mice (Taneja V., David C. S., Immunol Rev, Vol. 169:67-79(1999)).

An alternative also exists which relies on crossreactivity betweencertain human MHC molecules and particular MHC molecules expressed bylaboratory animals. Bertoni and colleagues (Bertoni et al., J Immunol,Vol. 161(8):4447-55 (1998)) have noted that appreciable crossreactivitycan be demonstrated between certain HLA Class I supertypes and certainPATR molecules expressed by chimpanzees. Crossreactivity between humanand macaques at the level of Class II (Geluk et al., J Exp Med, Vol.177(4):979-87 (1993)) and Class I molecules (Dzuris, et al., J.Immunol., July 1999) has also been noted. Finally, it can also be notedthat the motif recognized by human HLA B7 supertype is essentially thesame as the one recognized by the murine Class I L^(d) (Rammensee etal., Immunogenetics, Vol. 41(4):178-228 (1995)). Of relevance to testingHLA DR restricted epitopes in mice, it has been shown by Wall, et al(Wall et al., J. Immunol., 152:4526-36 (1994)) that similarities existin the motif of DR1 and IA^(b). We routinely breed our transgenic miceto take advantage of this fortuitous similarity. Furthermore, we havealso shown that most of our peptides bind to IA^(b), so that we usethese mice for the study of CTL and HTL immunogenicity.

Measuring and Quantitating Immune Responses from Clinical Samples

A crucial element to assess vaccine performance is to evaluate itscapacity to induce immune responses in vivo. Analyses of CTL and HTLresponses against the immunogen, as well as against common recallantigens are commonly used and are known in the art. Assays employedincluded chromium release, lymphokine secretion and lymphoproliferationassays.

More sensitive techniques such as the ELISPOT assay, intracellularcytokine staining, and tetramer staining have become available in theart. It is estimated that these newer methods are 10- to 100-fold moresensitive than the common CTL and HTL assays (Murali-Krishna et al.,Immunity, Vol. 8(2):177-87 (1998)), because the traditional methodsmeasure only the subset of T cells that can proliferate in vitro, andmay, in fact, be representative of only a fraction of the memory T cellcompartment (Ogg G. S., McMichael A. J., Curr Opin Immunol, Vol.10(4):393-6 (1998)). Specifically in the case of HIV, these techniqueshave been used to measure antigen-specific CTL responses from patientsthat would have been undetectable with previous techniques (Ogg et al.,Science, Vol. 279(5359):2103-6 (1998); Gray et al., J Immunol, Vol.162(3):1780-8 (1999); Ogg et al., J Virol, Vol. 73(11):9153-60 (1999);Kalams et al., J Virol, Vol. 73(8):6721-8 (1999); Larsson et al., AIDS,Vol. 13(7):767-77 (1999); Come et al., J Acquir Immune Defic Syndr HumRetrovirol, Vol. 20(5):442-7 (1999)).

With relatively few exceptions, direct activity of freshly isolatedcells has been difficult to demonstrate by the means of traditionalassays (Ogg G. S., McMichael A. J., Curr Opin Immunol, Vol. 10(4):393-6(1998)). However, the increased sensitivity of the newer techniques hasallowed investigators to detect responses from cells freshly isolatedfrom infected humans or experimental animals (Murali-Krishna et al.,Immunity, Vol. 8(2):177-87 (1998); Ogg G. S., McMichael A. J., Curr OpinImmunol, Vol. 10(4):393-6 (1998)). The availability of these sensitiveassays, which do not depend on an in vitro restimulation step, hasgreatly facilitated the study of CTL function in natural infection andcancer. In contrast, assays utilized as an endpoint to judgeeffectiveness of experimental vaccines are usually performed inconjunction with one or more in vitro restimulation steps (Ogg G. S.,McMichael A. J., Curr Opin Immunol, Vol. 10(4):393-6 (1998)). In fact,with few exceptions (Hanke et al., Vaccine, Vol. 16(4):426-35 (1998)),freshly isolated Class I-restricted CD8+ T cells have been difficult todemonstrate in response to immunization with experimental vaccinesdesigned to elicit CTL responses. The use of sensitive assays, such asELISPOT or in situ IFNγ ELISA, have been combined with a restimulationstep to achieve maximum sensitivity; MHC tetramers are also used forthis purpose.

MHC tetramers were first described in 1996 by Altman and colleagues.They produced soluble HLA-A2 Class I molecules that were folded withHIV-specific peptides containing a CTL epitope complexed together intotetramers tagged with fluorescent markers. These are used to labelpopulations of T cells from HIV-infected individuals that recognize theepitope (Ogg G. S., McMichael A. J., Curr Opin Immunol, Vol. 10(4):393-6(1998)). These cells were then quantified by flow cytometry, providing afrequency measurement for the T cells that are specific for the epitope.This technique has become very popular in HIV research as well as inother infectious diseases (Ogg G. S., McMichael A. J., Curr OpinImmunol, Vol. 10(4):393-6 (1998); Ogg et al., Science, Vol.279(5359):2103-6 (1998); Gray et al., J Immunol, Vol. 162(3):1780-8(1999); Ogg et al., J Virol, Vol. 73(11):9153-60 (1999); Kalams et al.,J Virol, Vol. 73(8):6721-8 (1999)). However, HLA polymorphism can limitthe general applicability of this technique, in that the tetramertechnology relies on defined HLA/peptide combinations. However, it hasbeen shown that a variety of peptides, including HIV-derived peptides,are recognized by peptide-specific CTL lines in the context of differentmembers of the A2, A3 and B7 supertypes (Threlkeld et al., J Immunol,Vol. 159(4):1648-57 (1997); Bertoni et al., J Clin Invest, Vol.100(3):503-13 (1997)). Taken together these observations demonstratethat a T cell receptor (TCR) for a given MHC/peptide combination canhave detectable affinity for the same peptide presented by a differentMHC molecule from the same supertype.

In circumstances in which efficacy of a prophylactic vaccine isprimarily correlated with the induction of a long-lasting memoryresponse, restimulation assays can be the most appropriate and sensitivemeasures to monitor vaccine-induced immunological responses. Conversely,in the case of therapeutic vaccines, the main immunological correlate ofactivity can be the induction of effector T cell function, most aptlymeasured by primary assays. Thus, the use of sensitive assays allows forthe most appropriate testing strategy for immunological monitoring ofvaccine efficacy.

Antigenicity of Multi-Epitope Constructs in Transfected Human APC's

Antigenicity assays are performed to evaluate epitope processing andpresentation in human cells. An episomal vector to efficiently transfecthuman target cells with multi-epitope nucleic acid vaccines is used toperform such an analysis.

For example, 221 A2 K^(b) target cells were transfected with an HIV-1epigene vaccine. The 221 A2 K^(b) target cell expresses the A2K^(b) genethat is expressed in HLA transgenic mice, but expresses no endogenousClass I (Shimizu Y, DeMars R., J Immunol, Vol. 142(9):3320-8 (1989)).These transfected cells were assayed for their capacity to presentantigen to CTL lines derived from HLA transgenic mice and specific forvarious HIV-derived CTL epitopes. To correct for differences in antigensensitivity of different CTL lines, peptide dose titrations, usinguntransfected cells as APC, were run in parallel. Representative data ispresented in FIG. 8. In the case of HIV Pol 498-specific CTL, thetransfected target cells induced the release of 378 pg/ml of IFNγ.Inspection of the peptide dose responses reveals that, 48 ng/ml ofexogenously added peptide was necessary to achieve similar levels ofIFNγ release. These results demonstrate that relatively large amounts ofPol 498 epitope are presented by the transfected cells, equivalent to 48ng/ml of exogenously added peptide.

TABLE 5 Comparison between antigenicity in transfected human cells andimmunogenicity in HLA transgenic mice of the HIV-1 minigene AntigenicityImmunogenicity Epitope Peptide Equivalents¹⁾ n²⁾ % response³⁾Magnitude⁴⁾ HIV Pol 498 30.5 (6) 95% 46.7 HIV Env 134 6.2 (3) 62% 16.1HIV Nef 221 2.1 (5) 82% 3.8 HIV Gag 271 <0.2 (6) 31% 4 ¹⁾ng/ml; ²⁾numberof independent experiments; ³⁾% of CTL cultures yielding positiveresults; ⁴⁾Lytic Units

By comparison, less than 25 pg/ml IFNγ was detected utilizing the CTLspecific for the Gag 271 epitope. The control peptide titration withuntransfected target cells revealed that this negative result could notbe ascribed to poor sensitivity of the particular CTL line utilized,because as little as 0.2 pg/ml of “peptide equivalents” (PE) could bedetected. Thus, it appears that the Gag 271 epitope is not efficientlyprocessed and presented in the HIV-1 transfected target cells. Utilizingthe “peptide equivalents” figure as an approximate quantitation of theefficiency of processing, it can be estimated that at least 200-foldless Gag 271 is presented by the transfected targets, compared to thePol 498 epitope.

The results of various independent determinations for four differentepitopes contained within HIV-FT are compiled in Table 5. The amount ofeach epitope produced from the HIV-FT transfected cells ranged from 30.5PE for Pol 498, to a low of less than 0.2 PE for Gag 271. The twoepitopes Env 134 and Nef 221 were associated with intermediate values,of 6.1 and 2.1 PE, respectively.

These results were next correlated with the in vivo immunogenicityvalues observed for each epitope after immunization with the HIV-FTconstruct. The Pol 498 epitope was also the most immunogenic, as wouldbe predicted. The Env 134 and Nef 221 epitopes, for which intermediateimmunogenicity was observed in vivo, were also processed in vitro withintermediate efficiency by the transfected human cells. Finally, the Gag271, for which no detectable in vitro processing was observed, was alsoassociated with in vivo immunogenicity suboptimal in both frequency andmagnitude.

These data have several important implications. First, they suggest thatdifferent epitopes contained within a given construct may be processedand presented with differential efficiency. Second, they suggest thatimmunogenicity is proportional to the amount of processed epitopegenerated. Finally, these results provide an important validation of theuse of transgenic mice for the purpose of optimization of multi-epitopevaccines destined for human use.

III. Preparation of Multi-Epitope Constructs

Epitopes for inclusion in the multi-epitope constructs typically bearHLA Class I or Class II binding motifs, as described for example in PCTapplications PCT/US00/27766, or PCT/US00/19774. Multi-epitope constructscan be prepared according to the methods set forth in Ishioka, et al.,J. Immunol. (1999) 162(7):3915-3925, for example.

Multiple HLA class II or class I epitopes present in a multi-epitopeconstruct can be derived from the same antigen, or from differentantigens. For example, a multi-epitope construct can contain one or moreHLA epitopes that can be derived from two different antigens of the samevirus or from two different antigens of different viruses. Epitopes forinclusion in a multi-epitope construct can be selected by one of skillin the art, e.g., by using a computer to select epitopes that containHLA allele-specific motifs or supermotifs. The multi-epitope constructsof the invention can also encode one or more broadly cross-reactivebinding, or universal, HLA class II epitopes, e.g., PADRE® epitope(Epimmune, San Diego, Calif.), (described, for example, in U.S. Pat.Nos. 5,736,142; 6,413,935; and 5,679,640) or a PADRE® family molecule.

Universal HLA Class II epitopes can be advantageously combined withother HLA Class I and Class II epitopes to increase the number of cellsthat are activated in response to a given antigen and provide broaderpopulation coverage of HLA-reactive alleles. Thus, the multi-epitopeconstructs of the invention can include HLA epitopes specific for anantigen, universal HLA class II epitopes, or a combination of specificHLA epitopes and at least one universal HLA class II epitope.

HLA Class I epitopes are generally less than about 15 residues inlength, preferably 13 residues or less in length and preferably areabout 8 to about 13 amino acids in length, more preferably about 8 toabout 11 amino acids in length (e.g. 8, 9, 10, or 11), and mostpreferably about 9 amino acids in length. HLA Class II epitopes aregenerally less than about 50 residues in length and usually consist ofabout 6 to about 30 residues, more usually between about 12 to 25, andoften about 15 to 20 residues, and can encode an epitope peptide ofabout 7 to about 23, preferably about 7 to about 17, more preferablyabout 11 to about 15 (e.g. 11, 12, 13, 14, or 15), and most preferablyabout 13 amino acids in length. An HLA Class I or II epitope can bederived from any desired antigen of interest. The antigen of interestcan be a viral antigen, surface receptor, tumor antigen, oncogene,enzyme, or any pathogen, cell or molecule for which an immune responseis desired. Epitopes can be selected based on their ability to bind oneor multiple HLA alleles. Epitopes that are analogs of naturallyoccurring sequences can also be included in the multi-epitope constructsdescribed herein. Such analog peptides are described, for example, inPCT applications PCT/US97/03778, PCT/US00/19774, and co-pending U.S.Ser. No. 09/260,714 filed Mar. 1, 1999.

Given the methods described herein for optimizing epitope configurationand spacers between the epitopes, the skilled artisan may include anyHLA epitopes into the multi-epitope constructs described herein. FIGS.2, 3, 9, 17, 18A-18N, 27A, 28A, 29A, and Tables 13, 14, 18, and 19depict exemplary multi-epitope constructs using epitopes listed in FIGS.19A-19E. Exemplary constructs are also set forth in FIGS. 20B, 20D, 20E,and 20F (epitopes are listed in FIG. 20A); FIGS. 21B, 21D, and 21E(epitopes are listed in FIG. 21A); FIGS. 22B, 22D, and 22E (epitopes arelisted in 22A); FIG. 23C; and FIGS. 24B and 24C (epitopes are listed inFIG. 24A). Multi-epitope constructs may include five or more, or six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,twenty, twenty-five, or thirty or more of the epitopes set forth inFIGS. 19A-19E, 20A, 21A, 22A, and 24A. Multi-epitope constructs thatinclude any combinations of these epitopes can be optimized using theprocedures set forth herein, and spacers can be optimized as well.

Multi-epitope constructs can be generated using methodology well knownin the art. For example, polypeptides comprising the multi-epitopeconstructs can be synthesized and linked. Typically, multi-epitopeconstructs are constructed using recombinant DNA technology.

IV. Expression Vectors and Construction of a Multi-Epitope Constructs

The multi-epitope constructs of the invention are typically provided asan expression vector comprising a nucleic acid encoding themulti-epitope polypeptide. Construction of such expression vectors isdescribed, for example in PCT/US99/10646. The expression vectors containat least one promoter element that is capable of expressing atranscription unit encoding the nucleic acid in the appropriate cells ofan organism so that the antigen is expressed and targeted to theappropriate HLA molecule. For example, for administration to a human, apromoter element that functions in a human cell is incorporated into theexpression vector.

In preferred embodiments, the invention utilizes routine techniques inthe field of recombinant genetics. Basic texts disclosing the generalmethods of use in this invention include Sambrook et al., MolecularCloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 1994); OligonucleotideSynthesis: A Practical Approach (Gait, ed., 1984); Kuijpers, NucleicAcids Research 18(17):5197 (1994); Dueholm, J. Org. Chem. 59:5767-5773(1994); Methods in Molecular Biology, volume 20 (Agrawal, ed.); andTijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, e.g., Part I, chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays” (1993)).

The nucleic acids encoding the epitopes are assembled in a constructaccording to standard techniques. In general, the nucleic acid sequencesencoding multi-epitope polypeptides are isolated using amplificationtechniques with oligonucleotide primers, or are chemically synthesized.Recombinant cloning techniques can also be used when appropriate.Oligonucleotide sequences are selected which either amplify (when usingPCR to assemble the construct) or encode (when using syntheticoligonucleotides to assemble the construct) the desired epitopes.

Amplification techniques using primers are typically used to amplify andisolate sequences encoding the epitopes of choice from DNA or RNA (seeU.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide toMethods and Applications (Innis et al., eds, 1990)). Methods such aspolymerase chain reaction (PCR) and ligase chain reaction (LCR) can beused to amplify epitope nucleic acid sequences directly from mRNA, fromcDNA, from genomic libraries or cDNA libraries. Restriction endonucleasesites can be incorporated into the primers. Multi-epitope constructsamplified by the PCR reaction can be purified from agarose gels andcloned into an appropriate vector.

Synthetic oligonucleotides can also be used to construct multi-epitopeconstructs. This method is performed using a series of overlappingoligonucleotides, representing both the sense and non-sense strands ofthe gene. These DNA fragments are then annealed, ligated and cloned.Oligonucleotides that are not commercially available can be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862(1981), using an automated synthesizer, as described in Van Devanter et.al., Nucleic Acids Res. 12:6159-6168 (1984). Purification ofoligonucleotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson & Reanier, J Chrom.255:137-149 (1983).

The epitopes of the multi-epitope constructs are typically subclonedinto an expression vector that contains a strong promoter to directtranscription, as well as other regulatory sequences such as enhancersand polyadenylation sites. Suitable promoters are well known in the artand described, e.g., in Sambrook et al. and Ausubel et al. Eukaryoticexpression systems for mammalian cells are well known in the art and arecommercially available. Such promoter elements include, for example,cytomegalovirus (CMV), Rous sarcoma virus long terminal repeats (RSVLTR) and Simmian Virus 40 (SV40).

The expression vector typically contains a transcription unit orexpression cassette that contains all the additional elements requiredfor the expression of the multi-epitope construct in host cells. Atypical expression cassette thus contains a promoter operably linked tothe multi-epitope construct and signals required for efficientpolyadenylation of the transcript. Additional elements of the cassettemay include enhancers and introns with functional splice donor andacceptor sites.

In addition to a promoter sequence, the expression cassette can alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic cells may beused. Expression vectors containing regulatory elements from eukaryoticviruses are typically used as eukaryotic expression vectors, e.g., SV40vectors, CMV vectors, papilloma virus vectors, and vectors derived fromEpstein Bar virus.

The multi-epitope constructs of the invention can be expressed from avariety of vectors including plasmid vectors as well as viral orbacterial vectors. Examples of viral expression vectors includeattenuated viral hosts, such as vaccinia or fowlpox. As an example ofthis approach, vaccinia virus is used as a vector to express nucleotidesequences that encode the peptides of the invention. Upon introductioninto a host bearing a tumor, the recombinant vaccinia virus expressesthe immunogenic peptide, and thereby elicits a host CTL and/or HTLresponse. Vaccinia vectors and methods useful in immunization protocolsare described in, e.g., U.S. Pat. No. 4,722,848.

A wide variety of other vectors useful for therapeutic administration orimmunization, e.g. adeno and adeno-associated virus vectors, retroviralvectors, non-viral vectors such as BCG (Bacille Calmette Guerin),Salmonella typhi vectors, detoxified anthrax toxin vectors, and thelike, will be apparent to those skilled in the art.

Immunogenicity and antigenicity of the multi-epitope constructs areevaluated as described herein.

Targeting Sequences

The expression vectors of the invention may encode one or more MHCepitopes operably linked to a MHC targeting sequence, and are referredto herein as “targeting nucleic acids” or “targeting sequences.” The useof a MHC targeting sequence enhances the immune response to an antigen,relative to delivery of antigen alone, by directing the peptide epitopeto the site of MHC molecule assembly and transport to the cell surface,thereby providing an increased number of MHC molecule-peptide epitopecomplexes available for binding to and activation of T cells.

MHC Class I targeting sequences can be used in the present invention,e.g., those sequences that target an MHC Class I epitope peptide to acytosolic pathway or to the endoplasmic reticulum (see, e.g., Rammenseeet al., Immunogenetics 41:178-228 (1995)). For example, the cytosolicpathway processes endogenous antigens that are expressed inside thecell. Although not wishing to be bound by any particular theory,cytosolic proteins are thought to be at least partially degraded by anendopeptidase activity of a proteasome and then transported to theendoplasmic reticulum by the TAP molecule (transporter associated withprocessing). In the endoplasmic reticulum, the antigen binds to MHCClass I molecules. Endoplasmic reticulum signal sequences bypass thecytosolic processing pathway and directly target endogenous antigens tothe endoplasmic reticulum, where proteolytic degradation into peptidefragments occurs. Such MHC Class I targeting sequences are well known inthe art, and include, e.g., signal sequences such as those from Igkappa, tissue plasminogen activator or insulin. A preferred signalpeptide is the human Ig kappa chain sequence. Endoplasmic reticulumsignal sequences can also be used to target MHC Class II epitopes to theendoplasmic reticulum, the site of MHC Class I molecule assembly. MHCClass II targeting sequences can also be used in the invention, e.g.,those that target a peptide to the endocytic pathway. These targetingsequences typically direct extracellular antigens to enter the endocyticpathway, which results in the antigen being transferred to the lysosomalcompartment where the antigen is proteolytically cleaved into antigenpeptides for binding to MHC Class II molecules. As with the normalprocessing of exogenous antigen, a sequence that directs a MHC Class IIepitope to the endosomes of the endocytic pathway and/or subsequently tolysosomes, where the MHC Class II epitope can bind to a MHC Class IImolecule, is a MHC Class II targeting sequence. For example, a group ofMHC Class II targeting sequences useful in the invention are lysosomaltargeting sequences, which localize polypeptides to lysosomes. Since MHCClass II molecules typically bind to antigen peptides derived fromproteolytic processing of endocytosed antigens in lysosomes, a lysosomaltargeting sequence can function as a MHC Class II targeting sequence.Lysosomal targeting sequences are well known in the art and includesequences found in the lysosomal proteins LAMP-1 and LAMP-2 as describedby August et al. (U.S. Pat. No. 5,633,234, issued May 27, 1997), whichis incorporated herein by reference.

Other lysosomal proteins that contain lysosomal targeting sequencesinclude HLA-DM. HLA-DM is an endosomal/lysosomal protein that functionsin facilitating binding of antigen peptides to MHC Class II molecules.Since it is located in the lysosome, HLA-DM has a lysosomal targetingsequence that can function as a MHC Class II molecule targeting sequence(Copier et al., J. Immunol. 157:1017-1027 (1996), which is incorporatedherein by reference).

The resident lysosomal protein HLA-DO can also function as a lysosomaltargeting sequence. In contrast to the resident lysosomal proteinsLAMP-1 and HLA-DM, which encode specific Tyr-containing motifs thattarget proteins to lysosomes, HLA-DO is targeted to lysosomes byassociation with HLA-DM (Liljedahl et al., EMBO J. 15:4817-4824 (1996)),which is incorporated herein by reference. Therefore, the sequences ofHLA-DO that cause association with HLA-DM and, consequently,translocation of HLA-DO to lysosomes can be used as MHC Class IItargeting sequences. Similarly, the murine homolog of HLA-DO, H2-DO, canbe used to derive a MHC Class II targeting sequence. A MHC Class IIepitope can be fused to HLA-DO or H2-DO and targeted to lysosomes.

In another example, the cytoplasmic domains of B cell receptor subunitsIg-α and Ig-β mediate antigen internalization and increase theefficiency of antigen presentation as described in, for example,Bonnerot et al., Immunity 3:335-347 (1995). Therefore, the cytoplasmicdomains of the Ig-α and Ig-β proteins can function as MHC Class IItargeting sequences that target a MHC Class II epitope to the endocyticpathway for processing and binding to MHC Class II molecules.

Another example of a MHC Class II targeting sequence that directs MHCClass II epitopes to the endocytic pathway is a sequence that directspolypeptides to be secreted, where the polypeptide can enter theendosomal pathway. These MHC Class II targeting sequences that directpolypeptides to be secreted mimic the normal pathway by which exogenous,extracellular antigens are processed into peptides that bind to MHCClass II molecules. Any signal sequence that functions to direct apolypeptide through the endoplasmic reticulum and ultimately to besecreted can function as a MHC Class II targeting sequence so long asthe secreted polypeptide can enter the endosomal/lysosomal pathway andbe cleaved into peptides that can bind to MHC Class II molecules.

In another example, the Ii protein binds to MHC Class II molecules inthe endoplasmic reticulum, where it functions to prevent peptidespresent in the endoplasmic reticulum from binding to the MHC Class IImolecules. Therefore, fusion of a MHC Class II epitope to the Ii proteintargets the MHC Class II epitope to the endoplasmic reticulum and a MHCClass II molecule. For example, the CLIP sequence of the Ii protein canbe removed and replaced with a MHC Class II epitope sequence so that theMHC Class II epitope is directed to the endoplasmic reticulum, where theepitope binds to a MHC Class II molecule.

In some cases, antigens themselves can serve as MHC Class II or Itargeting sequences and can be fused to a universal MHC Class II epitopeto stimulate an immune response. Although cytoplasmic viral antigens aregenerally processed and presented as complexes with MHC Class Imolecules, long-lived cytoplasmic proteins such as the influenza matrixprotein can enter the MHC Class II molecule processing pathway asdescribed in, for example, Guéguen & Long, Proc. Natl. Acad. Sci. USA93:14692-14697 (1996). Therefore, long-lived cytoplasmic proteins canfunction as a MHC Class I and/or MHC Class II targeting sequence. Forexample, an expression vector encoding influenza matrix protein fused toa universal MHC Class II epitope can be advantageously used to targetinfluenza antigen and the universal MHC Class II epitope to the MHCClass I and MHC Class II pathway for stimulating an immune response toinfluenza.

Other examples of antigens functioning as MHC Class II targetingsequences include polypeptides that spontaneously form particles. Thepolypeptides are secreted from the cell that produces them andspontaneously form particles, which are taken up into anantigen-presenting cell by endocytosis such as receptor-mediatedendocytosis or are engulfed by phagocytosis. The particles areproteolytically cleaved into antigen peptides after entering theendosomal/lysosomal pathway.

One such polypeptide that spontaneously forms particles is HBV surfaceantigen (HBV-S) as described in, for example, Diminsky et al., Vaccine15:637-647 (1997) or Le Borgne et al., Virology 240:304-315 (1998).Another polypeptide that spontaneously forms particles is HBV coreantigen as described in, for example, Kuhrober et al., InternationalImmunol. 9:1203-1212 (1997). Still another polypeptide thatspontaneously forms particles is the yeast Ty protein as described in,for example, Weber et al., Vaccine 13:831-834 (1995). For example, anexpression vector containing HBV-S antigen fused to a universal MHCClass II epitope can be advantageously used to target HBV-S antigen andthe universal MHC Class II epitope to the MHC Class II pathway forstimulating an immune response to HBV.

Administration In Vivo

The invention also provides methods for stimulating an immune responseby administering an expression vector of the invention to an individual.Administration of an expression vector of the invention for stimulatingan immune response is advantageous because the expression vectors of theinvention target MHC epitopes to MHC molecules, thus increasing thenumber of CTL and HTL activated by the antigens encoded by theexpression vector.

Initially, the expression vectors of the invention are screened in mouseto determine the expression vectors having optimal activity instimulating a desired immune response. Initial studies are thereforecarried out, where possible, with mouse genes of the MHC targetingsequences. Methods of determining the activity of the expression vectorsof the invention are well known in the art and include, for example, theuptake of ³H-thymidine to measure T cell activation and the release of⁵¹Cr to measure CTL activity as described below in Examples II and III.Experiments similar to those described in Example IV are performed todetermine the expression vectors having activity at stimulating animmune response. The expression vectors having activity are furthertested in human. To circumvent potential adverse immunological responsesto encoded mouse sequences, the expression vectors having activity aremodified so that the MHC Class I or MHC Class II targeting sequences arederived from human genes. For example, substitution of the analogousregions of the human homologs of genes containing various MHC Class I orMHC Class II targeting sequences are substituted into the expressionvectors of the invention. Expression vectors containing human MHC ClassI or MHC Class II targeting sequences, such as those described inExample I below, are tested for activity at stimulating an immuneresponse in human.

The invention also relates to pharmaceutical compositions comprising apharmaceutically acceptable carrier and an expression vector of theinvention. Pharmaceutically acceptable carriers are well known in theart and include aqueous or non-aqueous solutions, suspensions andemulsions, including physiologically buffered saline, alcohol/aqueoussolutions or other solvents or vehicles such as glycols, glycerol, oilssuch as olive oil or injectable organic esters.

A pharmaceutically acceptable carrier can contain physiologicallyacceptable compounds that act, for example, to stabilize the expressionvector or increase the absorption of the expression vector. Suchphysiologically acceptable compounds include, for example,carbohydrates, such as glucose, sucrose or dextrans, antioxidants suchas ascorbic acid or glutathione, chelating agents, low molecular weightpolypeptides, antimicrobial agents, inert gases or other stabilizers orexcipients. Expression vectors can additionally be complexed with othercomponents such as peptides, polypeptides and carbohydrates. Expressionvectors can also be complexed to particles or beads that can beadministered to an individual, for example, using a vaccine gun. Oneskilled in the art would know that the choice of a pharmaceuticallyacceptable carrier, including a physiologically acceptable compound,depends, for example, on the route of administration of the expressionvector.

The invention further relates to methods of administering apharmaceutical composition comprising an expression vector of theinvention to stimulate an immune response. The expression vectors areadministered by methods well known in the art as described in, forexample, Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997)); Felgneret al. (U.S. Pat. No. 5,580,859, issued Dec. 3, 1996); Felgner (U.S.Pat. No. 5,703,055, issued Dec. 30, 1997); and Carson et al. (U.S. Pat.No. 5,679,647, issued Oct. 21, 1997). In one embodiment, themulti-epitope construct is administered as naked nucleic acid.

A pharmaceutical composition comprising an expression vector of theinvention can be administered to stimulate an immune response in asubject by various routes including, for example, orally,intravaginally, rectally, or parenterally, such as intravenously,intramuscularly, subcutaneously, intraorbitally, intracapsularly,intraperitoneally, intracisternally or by passive or facilitatedabsorption through the skin using, for example, a skin patch ortransdermal iontophoresis, respectively. Furthermore, the compositioncan be administered by injection, intubation or topically, the latter ofwhich can be passive, for example, by direct application of an ointmentor powder, or active, for example, using a nasal spray or inhalant. Anexpression vector also can be administered as a topical spray, in whichcase one component of the composition is an appropriate propellant. Thepharmaceutical composition also can be incorporated, if desired, intoliposomes, microspheres or other polymer matrices as described in, forexample, Felgner et al., U.S. Pat. No. 5,703,055; Gregoriadis, LiposomeTechnology, Vols. I to III (2nd ed. 1993). Liposomes, for example, whichconsist of phospholipids or other lipids, are nontoxic, physiologicallyacceptable and metabolizable carriers that are relatively simple to makeand administer.

The expression vectors of the invention can be delivered to theinterstitial spaces of tissues of an animal body as described in, forexample, Felgner et al., U.S. Pat. Nos. 5,580,859 and 5,703,055.Administration of expression vectors of the invention to muscle is aparticularly effective method of administration, including intradermaland subcutaneous injections and transdermal administration. Transdermaladministration, such as by iontophoresis, is also an effective method todeliver expression vectors of the invention to muscle. Epidermaladministration of expression vectors of the invention can also beemployed. Epidermal administration involves mechanically or chemicallyirritating the outermost layer of epidermis to stimulate an immuneresponse to the irritant (Carson et al., U.S. Pat. No. 5,679,647).

Other effective methods of administering an expression vector of theinvention to stimulate an immune response include mucosal administrationas described in, for example, Carson et al., U.S. Pat. No. 5,679,647.For mucosal administration, the most effective method of administrationincludes intranasal administration of an appropriate aerosol containingthe expression vector and a pharmaceutical composition. Suppositoriesand topical preparations are also effective for delivery of expressionvectors to mucosal tissues of genital, vaginal and ocular sites.Additionally, expression vectors can be complexed to particles andadministered by a vaccine gun.

The dosage to be administered is dependent on the method ofadministration and will generally be between about 0.1 μg up to about200 μg. For example, the dosage can be from about 0.05 μg/kg to about 50mg/kg, in particular about 0.005-5 mg/kg. An effective dose can bedetermined, for example, by measuring the immune response afteradministration of an expression vector. For example, the production ofantibodies specific for the MHC Class II epitopes or MHC Class Iepitopes encoded by the expression vector can be measured by methodswell known in the art, including ELISA or other immunological assays. Inaddition, the activation of T helper cells or a CTL response can bemeasured by methods well known in the art including, for example, theuptake of ³H-thymidine to measure T cell activation and the release of⁵¹Cr to measure CTL activity (see Examples II and III below).

The pharmaceutical compositions comprising an expression vector of theinvention can be administered to mammals, particularly humans, forprophylactic or therapeutic purposes. Examples of diseases that can betreated or prevented using the expression vectors of the inventioninclude infection with HBV, HCV, HIV and CMV as well as prostate cancer,renal carcinoma, cervical carcinoma, lymphoma, condyloma acuminatum andacquired immunodeficiency syndrome (AIDS).

In therapeutic applications, the expression vectors of the invention areadministered to an individual already suffering from cancer, autoimmunedisease or infected with a virus. Those in the incubation phase or acutephase of the disease can be treated with expression vectors of theinvention, including those expressing all universal MHC Class IIepitopes, separately or in conjunction with other treatments, asappropriate.

In therapeutic and prophylactic applications, pharmaceuticalcompositions comprising expression vectors of the invention areadministered to a patient in an amount sufficient to elicit an effectiveimmune response to an antigen and to ameliorate the signs or symptoms ofa disease. The amount of expression vector to administer that issufficient to ameliorate the signs or symptoms of a disease is termed atherapeutically effective dose. The amount of expression vectorsufficient to achieve a therapeutically effective dose will depend onthe pharmaceutical composition comprising an expression vector of theinvention, the manner of administration, the state and severity of thedisease being treated, the weight and general state of health of thepatient and the judgment of the prescribing physician.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention. It is understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof are suggested to personsskilled in the art and are to be included within the spirit and purviewof this application and scope of the appended claims.

Examples 1-9 provide examples of assays for evaluating theimmunogenicity and antigenicity of multi-epitope constructs.

Example 1 Antigenicity Assays

High-affinity peptide-specific CTL lines can be generated fromsplenocytes of transgenic mice that have been primed with DNA,peptide/IFA, or lipopeptide. Briefly, splenocytes from transgenic miceare stimulated 0.1 μg/ml peptide and LPS blasts. Ten days after theinitial stimulation, and weekly thereafter, cells are restimulated withLPS blasts pulsed for 1 hour with 0.1 μg/ml peptide. CTL lines areassayed 5 days following restimulation in an in situ IFNγ ELISA asdescribed above. Alternatively, CTL lines that are derived from, e.g.,patients infected with the targeted pathogen or who have the targeteddisease, e.g., cancer, can be used. Specific CTL lines that are notavailable either from transgenic mice or from patients are generatedfrom PBMC of normal donors, drawing on the expertise in the art.

Target cells to be used in these assays are Jurkat or 0.221 cellstransfected with A2.1/K^(b), A11/K^(b), A1/K^(b), or B7/K^(b) for CTLlines derived from transgenic mice. All these cell lines are currentlyavailable to us (Epimmune Inc., San Diego, Calif.). In the case of humanCTL lines, 0.221 cells transfected with the appropriate human HLA alleleare utilized. We currently have 0.221 cells transfected with A2 and A1,and are generating A11, A24 and B7 transfectants. In an alternativeembodiment, if unforeseen problems arise in respect to target cells, LPSblasts and EBV-transformed lines are utilized for murine and human CTLlines, respectively.

To assay for antigenicity, serially diluted CTLs are incubated with 10⁵target cells and multiple peptide concentrations ranging from 1 to 10⁻⁶μg/ml. In addition, CTLs are also incubated with target cellstransfected with an episomal vector containing a multi-epitope constructof interest. Episomal vectors are known in the art.

The relative amount of peptide generated by natural processing withinthe multi-epitope nucleic acid-transfected APCs is quantitated asfollows. The amount of IFNγ generated by the CTL lines upon recognitionof the transfected target cells are recorded. The amount of syntheticpeptide necessary to yield the same amount of IFNγ are interpolated froma standard curve generated when the same CTL line is incubated inparallel with known concentrations of peptide.

Example 2 Mice, Immunizations and Cell Cultures

The derivation of the HLA-A2.1/K^(b) (Vitiello et al., J Exp Med, Vol.173(4):1007-15 (1991)) and A11/K^(b) (Alexander et al., J Immunol, Vol.159(10):4753-61 (1997)) transgenic mice used in this study has beendescribed. HLA B7 K^(b) transgenic mice are available in house (EpimmuneInc., San Diego, Calif.). HLA DR2, DR3 and DR4 transgenic mice areobtained from C. David (Mayo Clinic). Non-transgenic H-2^(b) mice arepurchased from Charles River Laboratories or other commercial vendors.Immunizations are performed as described in (Ishioka et al., J Immunol,Vol. 162(7):3915-25 (1999)). All cells are grown in culture mediumconsisting of RPMI 1640 medium with HEPES (Gibco Life Technologies)supplemented with 10% FBS, 4 mM L-glutamine, 50 μM 2-ME, 0.5 mM sodiumpyruvate, 100 μg/ml streptomycin and 100 U/ml penicillin.

HLA transgenic mice and antigenicity assays are used for the purpose oftesting and optimization CTL responses. The natural crossreactivitybetween HLA-DR and IA^(b) can also be exploited to test HTL responses.This evaluation provides an assessment of the antigenicity andimmunogenicity of multi-epitope constructs.

Example 3 Proliferation Assays

To assess the ability of HTL epitopes to induce an immune response,assays such as proliferation assays are often performed. For example,mouse CD4 T lymphocytes are immunomagnetically isolated from splenicsingle cell suspensions using DynaBeads Mouse CD4 (L3T4) (Dynal).Briefly, 2×10⁷ spleen cells are incubated with 5.6×10⁷ magnetic beadsfor 40 minutes at 4° C., and then washed 3 times. Magnetic beads aredetached using DetachaBead Mouse CD4 (Dynal). Isolated CD4 T lymphocytes(2×10⁵ cells/well) are cultured with 5×10⁵ irradiated (3500 rad)syngeneic spleen cells in triplicate in flat-bottom 96-well microtiterplates. Purified peptides are added to wells at a final concentration of20, 1, 0.05 and 0 μg/ml and cells are cultured for a total of 4 days.Approximately 14 hour before harvesting, 1 μCi of ³H-thymidine (ICN) isadded to each well. The wells are harvested onto Unifilter GF/B plates(Packard) using the Filtermate Harvester (Packard). ³H-Thymidineincorporation is determined by liquid scintillation counting using theTopCount™ microplate scintillation counter (Packard).

Example 4 ⁵¹Chromium Release Assay

This assay to measure CTL activity is well known in the art. The assayquantifies the lytic activity of the T cell population by measuring thepercent ⁵¹Cr released from a ⁵¹Cr-labeled target population (Brunner etal., Immunology, Vol. 14(2):181-96 (1968)). Data derived from thechromium release assay is usually expressed either as a CTLfrequency/10⁶ cell (limiting dilution analysis, LDA; (Current Protocolsin Immunology, Vol 1, John Wiley & Sons, Inc., USA 1991 Chapter 3;Manual of Clinical Laboratory Immunology, Fifth edition, ASM Press, 1997Section R), or by a less cumbersome quantitative assessment of bulk CTLactivity (lytic Units; LU assay). In a LU assay, the standard E:T ratioversus percent cytotoxicity data curves generated in a ⁵¹Cr-releaseassay are converted into lytic units (LU) per 10⁶ effector cells, with 1LU defined as the lytic activity required to achieve 30% lysis of targetcells (Wunderlick, J., Shearer, G., and Livingston, A. In: J. Coligan,A. Kruisbeek, D. Margulies, E. Shevach, and W. Strober (Eds.), CurrentProtocols in Immunology, Vol 1, “Assays for T cell function: inductionand measurement of cytotoxic T lymphocyte activity.” John Wiley & Sons,Inc., USA, p. 3.11.18). The LU calculation allows quantifying responsesand thus readily comparing different experimental values.

Example 5 In situ IFNγ ELISA

An in situ IFNγ ELISA assay has been developed and optimized for bothfreshly isolated and peptide-restimulated splenocytes (see, e.g.,McKinney et al., J. Immunol. Meth. 237 (1-2):105-117 (2000)). This assayis based on the ELISPOT assay, but utilizes a soluble chromagen, makingit readily adaptable to high-throughput analysis. In both the primaryand restimulation assays, this technique is more sensitive than either atraditional supernatant ELISA or the ⁵¹Cr-release assay, in thatresponses are observed in the in situ ELISA that are not detectable inthese other assays. On a per cell basis, the sensitivity of the in situELISA is approximately one IFNγ secreting cell/10⁴ plated cells.

96-well ELISA plates are coated with anti-IFNγ (rat anti-mouse IFNα MAb,Clone R4-6A2, Pharmingen) overnight at 4° C., and then blocked for 2hours at room temperature with 10% FBS in PBS. Serially diluted primarysplenocytes or CTLs are cultured for 20 hours with peptide and 10⁵Jurkat A2.1/K^(b) cells/well at 37° C. with 5% CO₂. The following day,the cells are washed out and the amount of IFNγ that had been secretedinto the wells is detected in a sandwich ELISA, using biotinylatedα-IFNγ (rat anti-mouse IFNγ mAb, Clone XMG1.2, Pharmingen) to detect thesecreted IFNγ. HRP-coupled strepavidin (Zymed) and TMB (ImmunoPure® TMBSubstrate Kit, Pierce) are used according to the manufacturer'sdirections for color development. The absorbance is read at 450 nm on aLabsystems Multiskan RC ELISA plate reader. In situ IFNγ ELISA data isevaluated in secretory units (SU), based on the number of cells thatsecrete 100 pg of IFNγ in response to a particular peptide, correctedfor the background amount of IFN in the absence of peptide.

Example 6 ELISPOT Assay

The ELISPOT assay quantifies the frequency of T cells specific for agiven peptide by measuring the capacity of individual cells to beinduced to produce and release specific lymphokines, usually IFNγ. Theincreased sensitivity of the ELISPOT assay has allowed investigators todetect responses from cells freshly isolated from infected humans orexperimental animals (Murali-Krishna et al., Immunity, Vol. 8(2):177-87(1998); Ogg et al., Science, Vol. 279(5359):2103-6 (1998)). The ELISPOTassays are conducted as described above for the IFNγ ELISA until thefinal steps, where ExtrAvidin-AP (Sigma, 1:500 dilution) is used inplace HRP-strepavidin. Color is developed using the substrate 5-BCIP(BioRad) according to the manufacturer's directions. Spots are countedusing a phase contrast microscope. Alternatively, spots are countedutilizing the Zeiss KS ELISPOT reader. In this case the BCIP/NBT (Zymed)substrate is used.

The ELISPOT assay is routinely utilized to quantitate immune responses.The spots can be manually counted, however, in a preferred mode, a KSELISPOT reader from Zeiss, a microscope-based system with softwarespecifically designed to recognize and count spots is used.

Example 7 Tetramer Staining

Tetramer staining is a flow cytometric technique that detectsepitope-specific human CD8⁺ T-lymphocytes based on the interactionbetween the peptide epitope, class I antigen and the T-cell receptorspecific for the epitope. This assay allows for the rapid quantitationof epitope specific human CD8⁺ T-lymphocytes in freshly isolated bloodsamples. MHC tetramers for various HIV peptide/HLA combinations can beobtained from, e.g., the NIH repository (Tetramer Core Facility:http://www.miaid.nih.gov/reposit/tetramer/index.html). To labelepitope-specific cells, 1×10⁶ PBMC in a 100 μl volume are incubated inthe dark for 40 minutes with 5 μg/ml of the appropriate tetramer plusmonoclonal antibodies that recognize human CD3 and CD8 (available indifferent fluorochrome-conjugated forms from commercial sourcesincluding PharMingen, San Diego, Calif. or BioSource, Camarillo,Calif.). The cells are washed and paraformaldehyde fixed prior toanalysis using a FACSan or FACSCalibur flow cytometer (Becton DickinsonImmunocytometry Systems, San Jose, Calif.). Sample data are analyzedusing CellQuest software.

Example 8 Assays from Clinical Samples

Various assays to evaluate the specific CD8⁺ CTL activity in frozen PBMCsamples from patients or volunteers can be used. ELISPOT, chromiumrelease, in situ IFNγ release, proliferation and tetramer assays are alluseful to evaluate responses from various experimental models, e.g.,those of murine and/or primate origin.

Experimental methods for the murine version of these assays aredescribed above, and these are adapted to human systems as described(Livingston et al, J Immunol, Vol. 159(3):1383-92 (1997); Heathcote etal., Hepatology, Vol. 30(2):531-6 (1999); Livingston et al., J Immunol,Vol. 162(5):3088-95 (1999)) and can be further adapted a recognized byone of ordinary skill in the art. Calculations on the amounts of frozenPBMC samples necessary to complete the assays are also described greaterdetail in Example 14.

Example 9 Transgenic Animals

Transgenic mice (HLA-A2.1/K^(b) H2^(b); HLA-A11/K^(b); HLA-B7/K^(b)) areimmunized intramuscularly in the anterior tibialis muscle orsubcutaneously in the base of the tail with doses up to 100 μg of DNA orpeptide in 10-100 μl volumes. DNA is formulated in saline, and peptidesin IFA. 11-21 days later, the animals are sacrificed using CO₂asphyxiation, their spleens removed and used as the source of cells forin vitro determination of CTL function. Typically, 3-6 mice perexperimental group are used. In addition, spleens from non-immunizedmice are used as a source of APC for restimulation of CTL cultures. Bothmales and females of 8-12 weeks of age are used.

Example 10 Demonstration of Simultaneous Induction of Responses AgainstMultiple CTL and HTL Epitopes

Construction and Testing of CTL Epitope Strings:

This example provides an example of testing multiple CTL and HTLepitopes. For example, epitope strings encompassing 10-12 different CTLepitopes under the control of a single promoter are synthesized andincorporated in a standard plasmid, pcDNA 3.1 (Invitrogen, San Diego).These constructs include a standard signal sequence and a universal HTLepitope, PADRE® epitope. Each set of epitopes is chosen to allowbalanced population coverage. To facilitate testing and optimization, abalanced representation of epitopes that have been shown to beimmunogenic in transgenic mice, and/or antigenic in humans are included.

The specific order of these CTL epitopes is chosen to minimize Class Ijunctional motifs by the use of the computer program, as describedherein. If, despite best efforts regarding order optimization, potentialjunctional epitopes are still present in a construct in accordance withthe invention, corresponding peptides are synthesized to monitor for CTLresponses against such epitopes in HLA transgenic mice. Generally,minimization of junctional motifs is successful and adequate. However,if responses against any junctional epitopes are detected, thesejunctional epitopes are disrupted by the use of short one to two residuespacers, such as K, AK, KA, KK, or A, compatible with expectedproteolytic cleavage preferences discussed in the previous sections.

Since the ultimate use of optimized constructs is a human vaccine,optimized human codons are utilized. Similarly, if such constructs wereto be expressed in bacteria or S19 cells, the codon utilization could bemodified to provide expression in these systems. However, to facilitatethe optimization process in HLA transgenic mice, care is applied toselect, whenever possible, human codons that are also optimal for mice.Human and murine codon usage is very similar. See, for example, Tables21 and 22.

Human cells transfected with the various multi-epitope nucleic acidvaccine constructs can be used in antigenicity assays, conducted inparallel with in vivo testing in HLA transgenic mice. Any potentialdiscrepancy between multi-epitope vaccine efficacy, due to thedifferential codon usage, is addressed by the availability of these twodifferent assay systems.

Typically, antigenicity and immunogenicity testing of plasmid constructsis conducted in parallel. In vivo testing in transgenic mice areperformed for A2, A11, and B7 HLA transgenic mice. Following a protocolwell established in our laboratory, cardiotoxin pretreated mice areinjected i.m. with 100 μg of each plasmid and responses evaluated elevendays later (Ishioka et al., J Immunol, Vol. 162(7):3915-25 (1999)).Assays will include ELISPOT from freshly isolated cells, as well asinterferon gamma release and cytotoxicity chromium release assays fromrestimulated cell cultures. All of the above mentioned techniques arewell established in the art. The simultaneous measurement of responsesagainst epitopes is not problematic, as large colonies of transgenicmice are already established “in house” for these HLA types. Groups offour to six mice are adequate to measure responses against six to tendifferent epitopes, in multiple readout assays. Testing of HLAA2-restricted, HIV-derived epitopes in HLA A2 transgenic mice istypically employed. However, should problems be encountered,antigenicity testing using human APC can be used as an alternativestrategy, or, can be used to complement the transgenic mice studies.

For the purpose of extending the correlation between immunogenicity intransgenic animals and antigenicity, as noted in the studies reportedherein, antigenicity testing is utilized to evaluate responses againstepitopes such as Pol 498, Env 134, Nef 221, Gag 271, for which highaffinity CTL lines are already available in house. For the purpose ofgenerating additional suitable CTL lines, direct immunization of HLAtransgenic mice with peptides emulsified in adjuvant, or lipopeptidesare utilized, as described herein, and routinely applied in ourlaboratory, to generate lines for use in antigenicity assays.

Antigenicity assays are also used, as a primary readout for epitopes forwhich in vivo optimization experiments are not feasible. These epitopesinclude A24 and possibly A1 restricted epitopes, as well as any epitopewhich is non-immunogenic in HLA transgenic mice. In any such cases, weuse human CTL lines, generated from pathogen-exposed individuals.Alternatively, we generate CTL lines for in vitro CTL induction, usingGMCSF/IL4-induced dendritic cells and peripheral blood lymphocytes(Celis et al., Proc Natl Acad Sci USA, Vol. 91(6):2105-9 (1994)).

Episomal vectors encoding the multi-epitope constructs are generated andtransfected into appropriate human cell lines to generate target cells.For example, the human T cell line Jurkat can be used, butlymphoblastoid cell lines have also been successfully utilized. Forexperiments utilizing CTL lines of human origin, well-characterizedHLA-matched, homozygous, EBV cell lines are commonly used as a source ofpurified-MHC and as target cells and are used as recipients of themulti-epitope nucleic acid transfections. For experiments utilizing CTLlines derived from HLA transgenic mice, a collection of Class Inegative, EBV-transformed, mutant cell lines 0.221 (Shimizu Y, DeMars R,J Immunol, Vol. 142(9):3320-8 (1989)) transfected with matchingHLA/K^(b) chimeric constructs are used as the recipient of themulti-epitope nucleic acid transfections. Such cells effectively presentpeptide antigens to CTL lines (Celis et al., Proc Natl Acad Sci USA,Vol. 91(6):2105-9 (1994)).

Construction and Testing of HTL Epitope Strings

Epitope strings encompassing 3-20 different HTL epitopes under thecontrol of a single promoter are synthesized and incorporated into astandard plasmid, pcDNA 3.1 (Invitrogen, San Diego). To facilitatetesting and optimization, each set of epitopes for a given construct ischosen to provide a balanced representation of epitopes which arealready known to be immunogenic in IA^(b) mice. In addition, all thepeptides corresponding to junctions are synthesized and tested forbinding to IA^(b) and, most importantly, for binding to a panel offourteen different DR molecules, representative of the most common DRalleles worldwide (Southwood et al., J Immunol, Vol. 160(7):3363-73(1998)). Thus, HTL epitopes that are not directed to an antigen ofinterest are not created within these plasmids. However, shouldjunctional regions with good DR binding potential (and hence, potentialDR restricted immunogenicity in vivo) be detected, a spacer such asGPGPG (SEQ ID NO:2) is introduced to eliminate them. In all constructs,the number of Class I junctional motifs will also be minimized, asdescribed herein.

Experimental vaccine plasmids are tested for immunogenicity using HLA DRtransgenic mice and/or mice of the H2b haplotype. Proliferation and/orcytokine production are measured (IL5, IFNγ). In a typical protocol,cardiotoxin treated mice are injected i.m. with 100 μg of each plasmid,and immune responses evaluated eleven days later (Ishioka et al., JImmunol, Vol. 162(7):3915-25 (1999)).

Testing for Interactions Between CTL and HTL Epitopes

The activities described above yield small, functional blocks ofepitopes, which are utilized to demonstrate simultaneousresponses/antigenicity against all epitopes analyzable. These blocks arethe subject to further optimization, as described in the next example.Using these same constructs, immunodominance is assessed. Specifically,all the CTL epitope constructs are mixed together, or all the HTLepitope constructs are mixed together. The results obtained with thepool of constructs are then compared with the results obtained with thesame construct, injected separately.

These constructs are also used to determine the effects of HTL epitopeson responses to CTL epitopes. Specifically, HTL and CTL containingplasmids are pooled and injected in mice, and CTL and HTL responses toselected epitopes are measured as described herein. Often, it isdetermined whether the presence, e.g., of HTL epitopes derived from thetarget antigen enhances CTL responses beyond the level of responseattained using a plasmid-containing a pan DR binding epitope, e.g.,PADRE® peptide or a PADRE® family molecule, in the CTL epitopeconstructs. Typically, it is also determined whether PADRE® peptideinhibits or augments responses to target antigen-derived HTL epitopes orconversely, if HTL epitopes derived from the antigen of interest inhibitor augment responses to PADRE® peptide.

Responses to a large number of epitopes are attainable using thismethodology. It is possible that the pooling of constructs may inhibitresponses against some of the weaker epitopes. In this case, the poolingexperiments are repeated after optimization.

Example 11 Optimization of CTL and HTL Multi-Epitope Constructs

This example describes the optimization the CTL and HTL constructsdescribed in Example 10. The potential influence of flanking residues onantigenicity and immunogenicity is also assessed in optimizing minigenconstructs. These studies involve the inclusion of flanking residues, asynonym for which is “spacers,” which have been designed to facilitateeffective processing.

Such an analysis can be performed as follows. First, the results oftesting of the CTL multi-epitope constructs described in Example 10 areanalyzed for trends and correlations between activity and the presenceof specific residues at the 3 residues flanking the epitope's N- andC-termini. Epitopes for which suboptimal CTL priming is noted, that aresuboptimal with respect to magnitude of response, are the targets forflanking region optimization. For each of the CTL multi-epitope nucleicacid vaccines, encoding 10-12 different CTL epitopes, ‘secondgeneration’ multi-epitope nucleic acid vaccines, with optimizedconfiguration, are produced.

In one embodiment, the first optimization design is to introduce eitheran Alanine (A) or Lysine (K) residue at position C+1 for all epitopesassociated with suboptimal performance. A second optimization design isto introduce in the C+1 position, the residue naturally occurring in thetarget antigen, e.g., HIV, that are associated with antigenicity.

For selected epitopes, additional modifications are also introduced.Specifically, the effect of introducing other residue spacers at theepitope C- and N-termini are also investigated. Depending on the resultsof the analysis of the multi-epitope nucleic acid vaccines described inExample 10, residues investigated may further include, for example, G,Q, W, S and T. If junctional epitopes are created by thesemodifications, then alternative epitope orders are rationally designedas described herein on order to eliminate the junctional epitopes. Allsecond-generation constructs are tested for antigenicity andimmunogenicity, as described herein.

As a result of these modifications, variations in activity thatcorrespond to specific modifications of the multi-epitope constructs areidentified. Certain modifications are found that have general,beneficial effects. To confirm this, generation and testing ofadditional multi-epitope nucleic acid vaccines in which all epitopes(also the ones which displayed acceptable antigenicity andimmunogenicity) are subject to the same modification are conducted. Insome instances, increased activity is noted for some epitopes but notothers, or less desirably that certain modifications increase theactivity of some, but decrease the activity of other epitopes. In suchcases, additional multi-epitope nucleic acid vaccines are designed andtested, to retain the beneficial modifications, while excluding thosealterations that proved to be detrimental or have no effect.

These multi-epitope nucleic acid vaccines are designated so that: a) aminimum of predicted junctional epitopes are present; and b) theepitopes which were not functional in the previous multi-epitope nucleicacid vaccines are now in a more efficacious context.

For HTL multi-epitope constructs, the data obtained from the “firstgeneration” constructs are inspected for trends, in terms of junctionalepitopes, and epitope position within the constructs, and proximity tospacers, e.g. GPGPG (SEQ ID NO:2) spacers. If specific trends aredetected, second generation constructs are designed based on thesetrends. Alternatively, in case of multi-epitope constructs yieldingsuboptimal activity, the potential effectiveness of other targetingstrategies, such as the ones based on Ii and LAMP are reevaluated, andcompared to no targeting and simple, leader sequence targeting.

When large variations in activity of either the CTL or HTL multi-epitopeconstructs described in this section are detected, the results areconsistent with influences such as conformational or “long-range”effects impacting construct activity. These variables can be analyzed bymeans of current molecular and cellular biology techniques. For example,cell lines transfected with the various multi-epitope constructs couldbe analyzed for mRNA expression levels, and stability by Northernanalysis or primer extension assays (Current Protocols in MolecularBiology, Vol 3, John Wiley & Sons, Inc. USA 1999).

In all multi-epitope nucleic acid vaccines, an antibody tag such asMYC/his can also be included. This tag allows for testing of proteinexpression levels. The inclusion of MYC/his tag (Manstein et al., Gene,Vol. 162(1):129-34 (1995)) also allows determination of the stability ofthe expressed products, by pulse-chase experiments. The results of theseassays can then be compared with the results of the antigenicity andimmunogenicity experiments. The results are inspected for the presenceof trends and general rules, and correlation between the differentvariables examined.

Example 12 Determination of the Simplest Plasmid Configuration Capableof Effective Delivery of Selected Epitopes

The experiments described in Examples 11 and 12 are designed to addressvariables concerning multi-epitope nucleic acid vaccine design. Ideally,a vector that can be used in humans is used through the entire program,but one DNA vaccine plasmid for the vaccine epitope optimization studiescan be used and then switched to a vector suitable for human use. Actualvector selection is dependent on several variables. For example, theavailability of vectors, suitable for human use, through a reliablesource, such as the National Gene Vector Laboratory (University ofMichigan) is a factor.

In this example, the optimized constructs are also ligated to formlarger blocks of epitopes. All constructs are preferably designed toincorporate PADRE® peptides and leader sequence targeting in the case ofCTL multi-epitope constructs. Specifically, two pairs of the 10-12 CTLepitope constructs are ligated to generate two 20-24 CTL epitopeconstructs. In a situation where ligation of epitopes yields suboptimal(decreased) activity compared to the smaller constructs, alternativecombinations and orders of ligation are investigated. The specific pairof 20-24 CTL epitope constructs yielding optimal activity are thenligated and the resulting construct encompassing all CTL epitopesevaluated for activity. Once again up to two alternative orientationsare investigated. Because of the relatively large size of thisconstruct, the specific effect of targeting sequences are confirmed,since it is possible that leader sequence targeting are more effectiveon small size constructs, while larger size constructs may be mosteffectively targeted by ubiquitin signals. Specifically, one constructwithout any specific targeting sequences is generated and compared to aconstruct that is targeted for degradation by the addition of aubiquitin molecule.

A similar strategy is used for HTL. Two pairs of the 3-5 HTL epitopeconstructs are ligated to generate two 7-9 HTL epitope constructs. Onceagain, in a situation where ligation of these epitopes yields suboptimal(decreased) activity, alternative combinations and order of ligation areinvestigated. The specific pair of 7-9 CTL epitope constructs yieldingoptimal activity are ligated and the resulting construct, encompassingall HTL epitopes, is evaluated for activity. Once again, up to twoalternative orientations are investigated.

Based on these results an optimized plasmid configuration capable ofeffective delivery of a panel, e.g., of HIV epitopes, is selected forclinical trial evaluation. Of course, epitopes from any antigen ofinterest (infectious or disease-associated) can be used alone or incombination. This configuration will entail one or more HTL epitopeconstructs and one or more CTL epitope constructs. A combination of onelong CTL and one long HTL epitope construct capable of effectivelydelivering all encoded epitopes, is most preferable, as it simplifiesfurther clinical development of the vaccine. In case undesirableinteractions between the two constructs are observed when co-injected,injection of the different plasmids in the same animals, but indifferent injection sites, or at different points in time can beexamined. Alternatively, if a single CTL construct and HTL constructencoding all the desired epitopes is not identified, pools of constructsare considered for further development.

Example 13 Evaluation and Characterization of CD8+ Lymphocyte ResponsesInduce Following Immunization with Multi-Epitope Vaccine

CD8+ lymphocyte responses were measured mostly relying on the ELISPOTtechnique. The ELISPOT assay is known in the art, and is regularly usedin our laboratory. An automated Zeiss ELISPOT reader is also used as setforth herein. The assays utilized to measure CD8+ responses areprimarily the IFNγ ELISPOT assay on freshly isolated cells as well ascells restimulated in vitro with peptide. In addition, in selectedinstances, chromium release assays are utilized. The results werecorrelated with the ones observed in the case of the ELISPOT assays.Tetramer staining on selected peptide/MHC combinations was alsoperformed.

The clinical assay was developed and validated. The timing of thisactivity coincides with the period of time that follows selection of aclinical vaccine epigene construct, and precedes the availability ofactual samples from individuals enrolled in the clinical trial. Assaysfor CTL evaluation can be established based on experience in the art,for example, experience in establishing assays for CTL evaluations inthe Phase I and II trials of an experimental HBV vaccine (Livingston etal, J Immunol, Vol. 159(3):1383-92 (1997); Heathcote et al., Hepatology,Vol. 30(2):531-6 (1999); Livingston et al., J Immunol, Vol.162(5):3088-95 (1999)). Specifically, Ficoll-purified PBMC derived fromnormal subjects, as well from, e.g., unvaccinated volunteers can beused. As noted previously, other antigenic target(s) can be used inaccordance with the invention.

Example 14 Design of Optimized Multi-Epitope DNA-Based VaccineConstructs

Optimized constructs were designed with the aid of the computer-assistedmethods described above which simultaneously minimize the formation ofjunctional epitopes and optimize C+1 processing efficiency. Thefollowing motifs were utilized for junctional minimization: murine K^(b)(XXXX(F or Y)X₂₋₃(L, I, M or V)); D^(b) (XXXXNX₂₋₃L, I, M or V)); humanA2 (X(L or M)X₆₋₇V); human A3/A11 (X(L, I, M or V)X₆₋₇(K, R or Y)); andhuman B7 (XPX₆₋₇(L, I, M, V or F)). The C+1 propensity values werecalculated from the data presented in FIG. 6 and are as follows: K=2.2;N=2; G=1.8; T=1.5; A,F,S=1.33; W,Q=1.2; R=1.7; M,Y=1; I=0.86; L=0.76;V,D,H,E,P=0. Insertion of up to four amino acids was permitted. Examplesof constructs designed by this procedure and other procedures set forthherein are depicted in FIG. 18. A number of these constructs werecharacterized in vitro and in vivo immunogenicity studies, which are setforth hereafter. FIG. 19 lists amino acid epitope sequences encoded bycertain nucleic acid sequences in the multi-epitope constructs.

Example 15 Immunogenicity Testing of Multi-Epitope CTL Constructs andInfluence of Flanking Amino Acids

HLA transgenic mice were used for immunogenicity testing of differentmulti-epitope constructs. One group of mice were pretreated by injecting50 μl of 10 μM cardiotoxin bilaterally into the tibialis anteriormuscle, and then four or five days later, 100 μg of a DNA constructdiluted in PBS was administered to the same muscle. In another group,each mouse was injected with a peptide emulsified in CFA, wherein thepeptide corresponds to an epitope within the DNA construct administeredto mice in the DNA injection group. Eleven to fourteen days afterimmunization, splenocytes from DNA vaccinated animals and peptidevaccinated animals were recovered and CTL activity was measured in oneof several assays, including a standard ⁵¹Cr-release assay, an ELISPOTassay that measured γ-IFN production by purified CD8+ T-lymphocyteswithout peptide epitope-specific restimulation, and an in situ ELISA,which included an in vitro epitope-specific restimulation step with apeptide epitope. Examples of CTL activity induced by the EP-HIV-1090construct upon stimulation with peptide epitopes are shown in FIG. 14A,and CTL activity induced by the PFCTL.1, PFCTL.2, and PFCTL.3 constructsupon stimulation with peptide epitopes are shown in FIG. 14B.

The effect of different amino acids in the C+1 flanking position wasdirectly evaluated by inserting different amino acids at the C+1position relative to the Core 18 epitope in the HBV.1 construct. Theimmunogenicity data clearly demonstrate reduced immunogenicity of theCore 18 epitope when it was flanked at the C+1 position by W, Y, or L(FIG. 6 b). In contrast, insertion of a single K residue dramaticallyincreased the CTL response to Core 18. Enhancement of CTL responses wasalso observed using R, C, N, or G at the C+1 position. These dataclearly demonstrate that C+1 processing optimization can improvemulti-epitope construct design.

Example 16 Immunogenicity Testing of Multi-Epitope HTL Constructs andInfluence of Spacer Sequences

A universal spacer consisting of GPGPG (SEQ ID NO:2) was developed toseparate HTL epitopes, thus disrupting junctional epitopes. The logicbehind the design of this spacer is that neither G nor P are used asprimary anchors, positions 1 and 6 in the core region of an HTL peptideepitope, by any known murine or human MHC Class I or MHC Class IImolecule. The gap of five amino acids introduced by this spacerseparates adjacent epitopes so the amino acids of two epitopes cannotphysically serve as anchors in the 1 and 6 positions. The utility of theGPGPG (SEQ ID NO:2) spacer was tested using synthetic peptides composedof four HIV-1 epitopes, one having three spacers and the other lackingspacers, known to bind mouse IA^(b). HIV 75mer was the construct havingthree GPGPG (SEQ ID NO:2) spacers and HIV 60mer was the constructlacking the three spacers. Immunization of CB6F1 mice with the peptidein CFA induced HTL responses against 3 of 4 of the epitopes in theabsence of the spacer but all epitopes were immunogenic when the spacerwas present (FIG. 15). This evidence demonstrates that spacers canimprove the performance of multi-epitope constructs.

The ability of multi-epitope HTL DNA-based constructs to induce an HTLresponse in vivo was evaluated by intramuscular immunization of H2^(bxd)mice with an EP-HIV-1043-PADRE® construct. The EP-HIV-1043-PADRE®construct is set forth in FIG. 18, and the difference between theEP-HIV-1043-PADRE® construct and EP-HIV-1043 is that the former includesa C-terminal GPGPG (SEQ ID NO:2) spacer followed by the PADRE® sequenceAKXVAAWTLKAAA (SEQ ID NO:1). Eleven days after immunization, no boosterimmunizations were administered, CD4 T cells were purified from thespleen, and peptide specific HTL responses were measured in a primaryγ-IFN ELISPOT assay. Examples of HTL activity induced by constructsencoding HIV epitopes are shown in FIG. 16. Overall, the HTL responsesinduced by DNA immunization with the multi-epitope HIV HTL constructwere generally of equal or greater magnitude than the responses inducedby peptide immunization.

Example 17 Development of an Epitope-Based HBV Immunotherapeutic Vaccine

1. Introduction

Natural Correlates of Viral Clearance

The cellular immune response associated with the natural clearance ofacute HBV infection is broad and multi-specific. This response includesboth CTL and HTL directed against epitopes from multiple viral geneproducts (Chisari, F. V. and Ferrari, C. Annu. Rev. Immunol. 13:29-60(1995)). Chronic HBV infection is rarely resolved by the immune system,but when this happens, viral clearance is associated with increases inCTL activity, ALT flares and reductions in viral load (Guidotti, L. G.and Chisari, F. V., Annu. Rev. Immunol. 19:65-91 (2001)). Viralclearance can also be induced in a significant fraction (10-15%) ofindividuals receiving IFN-α treatment and, similar to spontaneousclearance, the effect is correlated with increased cellular immuneresponses.

The magnitude of cellular immune responses associated with control ofHBV infection was investigated in several studies. For comparativepurposes, the following values (mean and range) represent the number ofantigen-specific cells per million CD8+ cells. Lohr and coworkersutilized ELISPOT assays to quantitate HBV-specific responses detected inperipheral blood lymphocytes (PBL) during the acute phase of infection(Lohr, H. F. et al., Liver 18:405-413 (1998)). They reported a range of400-2800 Spot Forming Cells (SFC) (mean 1400) responding to HBV core18-27. Maini et al. used tetramer staining, which is reported to beapproximately four-fold more sensitive than ELISPOT assays (Tan, L. C.et al., J. Immunol. 162:1827-1835 (1999)), and determined a range of80-14,000 tetramer-positive cells for the core 18-27 epitope, with amean of 4,000 (Maini, M. K. et al., Gastroenterology 117:1386-1396(1999)). Taking into account the differential sensitivity of the assays,this translates to an estimated range of 20 to 3500 ELISPOT-positivecells, with a mean of a 1000 specific cells.

Using the same assay, Webster et al., reported 7000 tetramer-positivecells for the core 18-27 epitope (1750 ELISPOT-positive cells), 200cells for the env 335 (50 ELISPOT-positive cells) and 1200 for the pol562 epitope ELISPOT-positive cells) (Webster, G. J. et al., Hepatology.32:1117-1124 (2000)). In the case of two other epitopes analyzed, a meanof 200 tetramer-positive cells (80-6000 range) for env 335, and a meanof 220 cells for the pol 562 epitope (80-3200 range) were observed(Maini, M. K. et al., Gastroenterology 117:1386-1396 (1999)). Roughestimates of these responses in terms of ELISPOT cells are a mean of 50SFC for env 335 (20-1500 range) and a mean of 55 SFC for pol 562 (20-800range). These data are comparable to data obtained utilizing the LDAassay, which is approximately 40-50-fold less sensitive than the ELISPOTassay (Murali-Krishna, K. et al., Adv. Exp. Med. Biol. 452:123-142(1998)). For example, Rehermann and colleagues estimated 15 cells werespecific for env 335, and 18 cells were specific for pol 445 (Rehermann,B. et al., J. Clin. Invest. 97:1655-1665 (1996)). Assuming a 45-folddifferential sensitivity of the assays, these values correspond to 675and 810 epitope-specific ELISPOT positive cells, respectively.Additional data comes from Lohr et al. who used ELISPOT assays toquantitate HBV-specific responses in patients that responded to IFN-αtreatment that resulted in viral clearance (Lohr H. F. et al., Liver18:4-5-413 (1998)). In this study, a mean of 600 SFC (range 200-1300)specific for HBV core 18-27 was reported.

In summary, CTL specific for various HBV epitopes are detected in PBLduring clearance of the HBV virus. The frequency of functional cellsdetected by ELISPOT ranged from 20-400 cells/million CD8+ cells (low) to820-3500 SFC/million CD8+ cells (high), with an average response between50-1000 SFC/million CD8+ cells.

The importance of HBV-specific CTL was demonstrated directly usingHBV-transgenic mice. Specifically, adoptive transfer of cloned CTLspecific for different viral antigens, including the env, core and polantigens, and restricted by murine MHC molecules, led to the eliminationof the expression of viral antigens (Tsui, L. V. et al., Proc. Natl.Acad. Sci. USA. 92:12398-12402 (1995); Guidotti, L. G. et al., Immunity.4:25-36 (1996)). These data clearly document the importance of CTLresponses to the control of HBV infection

The magnitude of HTL responses during HBV infection is generally lowerthan for CTL. Utilizing whole antigens and ELISPOT assays, Lohr et al.observed overall frequencies of 47±5.2 SFC per million CD4+ cells inpatients responding to IFN-α treatment and 42±12 SFC per million CD4+cells during acute infection. (Lohr, H. F. et al., Liver 18:405-413(1998)) Webster et al. reported that 2,900 tetramer-positive cells permillion CD4+ cells were detected against core antigen in a patient 10weeks post-infection (Webster, G. J. et al., Hepatology. 32:1117-1124(2000)).

In conclusion, these data provide a means of establishing a level ofimmunogenicity for therapeutic HBV vaccines designed to induce CTLresponses.

B. Immune Tolerance is Associated with Chronic HBV Infection

HBV epitope-specific immune tolerance is associated with chronic HBVinfection (Chisari, F. V. and Ferrari, C. Annu. Rev. Immunol. 13:29-60(1995); Alexander, J. et al., Immunol. Res. 18:79-92 (1998); Milich, D.R., Can. J. Gastroenterol. 14:781-787 (2000); Hilleman, M. R. et al.,Vaccine. 19:1837-1848 (2001); Jung, M. C. et al., Lancet Infect. Dis.2:43-50 (2002)). In the infected individual, high levels of viremia arebelieved to be responsible for this immune tolerant status. Althoughthis effect can be so pronounced that it leads to a generalized Th1/Th2imbalance and general peripheral tolerance, it does not result indeletion of HBV-specific CTL precursors (Rossol, S. et al., B. J. Clin.Invest. 99:3025-3033 (1997); Chen et al, Immunity 12: 83-93 (2000);Sette, A. D. et al., J. Immunol. 166:1389-1397 (2001)). Indeed, studiesin HBV-transgenic mice were used to demonstrate that tolerance can be“broken” by the use of epitope-based vaccines and non-pathogen derived,optimized HTL epitopes (Livingston, B. D. et al., J. Immunol.159:1383-1392 (1997); Alexander, J. et al., Immunol. Res. 18:79-92(1998); Sette, A. D. et al., J. Immunol. 166:1389-1397 (2001)). The datagenerated using patient samples obtained during spontaneous resolutionof HBV infection and during response to IFN-α treatment also suggeststhat this defect is reversible. Additional data to support thishypothesis was derived in studies utilizing the antiviral drug,lamivudine, as discussed below.

Previous HBV Immunotherapy Clinical Trials

Clinical studies using a lipopeptide vaccine composed of a promiscuousHTL epitope and the HBV core 18 CTL epitope, provided data to documentimmunogenicity of individual epitopes in normal volunteers (Livingston,B. D. et al., Hum. Immunol. 60:1013-1017 (1999); Livingston, B. D. etal., J. Immunol. 159:1383-1392 (1997); Vitiello, A. et al., J. Clin.Invest. 95:341-349 (1995)). The levels of CTL induced in healthysubjects were comparable to those measured in acutely infectedindividuals who clear the virus, either spontaneously or as a result ofIFN-α treatment. Subsequent trials in chronic HBV patients were,however, disappointing: The levels of CTL induced in these patients weresignificantly lower than the levels observed in normal subjects and noreductions in viral loads were observed. Importantly, at the time ofthese clinical trials, antiviral drug therapy was not available. Thus,there was no way to reduce the viremia associated with immune tolerance.

D. Effects of Antiviral Drug Therapy on HBV Replication, Integration andImmune System Tolerance

Chronic HBV infection is associated with high levels of viremiaaveraging about 2.2×10¹¹ viral particles per 3 liters of serum, which isequivalent to the average total body burden (Nowak, M. A. et al., Proc.Natl. Acad. Sci. USA. 93:4398-4402 (1996)). The presence of high numbersof viral particles in the serum is thought to be responsible, at leastin part, for the immune tolerance detected in chronic HBV patients(Schlaak, J. F. et al., J. Hepatology 30:353-358 (1999)). The nucleosideanalog lamivudine (Epivir-HBV) (GlaxoSmithKline, Research Triangle Park,N.C. 27709) is a reverse transcriptase inhibitor originally developedfor the treatment of HIV. It was also approved for the treatment ofchronic HBV infection, is known to have potent inhibitory effects on HBVreplication, and rapidly reduces the production of new infectious virusparticles in patients (Nowak, M. A. et al., Proc. Natl. Acad. Sci. USA.93:4398-4402 (1996)). In multiple studies, HBV DNA becomes undetectableduring lamivudine treatment in the majority of patients (Dienstag, J. L.et al., Hepatology 30:1082-1087 (1999); Boni, C. et al, Hepatology.33:963-971 (2001)). Within the first six months of treatment there is amajor decline in the level of viremia, which continues with longer-termtreatment. HBsAg and HBeAg levels decline over time in most patientsalthough the rate and magnitude are less than that observed for viralparticles. Liver enzymes also fall to near normal levels in the majorityof patients with 6 months or more of lamivudine therapy (Dienstag, J. L.et al., Hepatology 30:1082-1087 (1999); Boni, C. et al., Hepatology.33:963-971 (2001)). Lamivudine does not totally suppress viral proteinproduction because covalently closed-circular DNA (cccDNA) andintegrated HBV DNA will support the production of some viral proteinsover a prolonged period of time.

In addition, the hypo-responsiveness of HBV-specific CTL and HTL,typical of chronic HBV infection, appears to be overcome or at leastdecreased by lamivudine treatment (Boni, C. et al., B. J. Clin. Invest.102:968-975 (1998); Boni, C. et al., Hepatology. 33:963-971 (2001)).Interestingly, the rebound in T-cell activity appears as early as onemonth after initiation of lamivudine therapy following the initial sharpdecline in viremia. However, when lamivudine treatment is suspended,viral replication rebounds in as little as one week, depending on theduration of treatment (Dienstag, J. L. et al., N. Eng. J. Med.333:1657-1661 (1995); Dienstag, J. L. et al., Hepatology 30:1082-1087(1999)). Also, there has been reported a rapid emergence ofdrug-resistant HBV mutants. Thus, lamivudine alone is limited inusefulness as a therapy for chronic HBV infection.

E. Immunotherapeutic Vaccine Design

The design and evaluation of therapeutic vaccines capable of inducingcellular immune responses of the magnitude needed to control HBVreplication and ultimately, mediate viral clearance is of great clinicalimportance. Vaccines are designed to induce both HBV-specific CTL andHTL responses, and are tested clinically in both healthy volunteers andchronically-infected patients. In the latter group, patients arerestricted to those treated successfully with lamivudine or similarantiviral for a minimum of six months.

Epitope Selection

CTL Epitopes from the HLA-A2, -A3 and -B7 Supertype Families

The majority of HLA class I molecules can be classified into relativelyfew major HLA class I supertypes when grouped by the characteristics oftheir overlapping, yet independent, peptide binding repertoires (Table6A-B). By selecting epitopes capable of binding most, or all, of the HLAmolecules in a given supertype, it is possible to limit the numbers ofepitopes needed to produce an effective multi-epitope vaccine. Selectionof the most common HLA supertypes facilitates design of a vaccine fortreatment of individuals with HBV infection (Bertoni, R., J. et al., J.Clin. Invest. 100:503-513 (1997); Sette, A. et al., Immunogenetics.50:201-212 (1999); Sette, A. et al., Curr. Opin. Immunol. 10:478-482(1998)).

TABLE 6A Phenotypic frequencies of HLA Class I Phenotypic Frequency (%)Supertype HLA allele Asian Black E Cauc NA Cauc A2 A*0201 15.8 19.6 45.132.0 A*0202 0.2 8.7 1.5 3.7 A*0203 8.7 0.2 0.2 4.1 A*0206 10.8 0.6 0.27.8 A*6802 0.2 9.6 1.3 2.2 A3 A*0301 1.3 14.6 26.8 25.9 A*1101 35.3 1.111.5 12.4 A*3101 8.2 1.3 5.1 4.5 A*3301 5.2 4.0 1.8 1.6 A*6801 0.5 7.06.0 5.0 A1 A*0101 1.5 7.0 30.7 29.4 A*2902 0.5 5.1 6.3 5.7 A*3002 2.230.7 4.7 4.9 A24 A*2402 49.5 4.2 16.5 15.6 A*2301 0.2 17.9 3.2 4.5A*2902 0.5 5.1 6.3 5.7 A*3002 2.2 30.7 4.7 4.9 B7 B*0702 5.6 13.8 24.925.6 B*3501 9.3 9.0 16.0 17.4 B*5101 12.2 4.6 10.7 9.3 B*5301 0.2 19.40.6 1.2 B*5401 8.6 0.1 0.1 0.1

TABLE 6B Phenotypic frequencies of HLA Class II Phenotypic frequency (%)Antigen Asian Black E Cauc NA Cauc DR1 6.0 13.1 19.3 22.5 DR2w2 B1 34.729.2 27.6 27.3 DR3 5.2 22.4 24.7 21.0 DR4w4 0.9 3.3 14.3 14.8 DR4w14 1.70.7 4.3 7.5 DR4w15 16.0 1.0 1.5 1.7 DR5w11 7.7 23.1 18.2 18.5 DR6w1910.5 39.9 21.6 22.0 DR7 4.2 14.8 25.5 23.4 DR8w2 18.6 10.7 5.4 6.7 DR923.5 3.9 2.0 2.0 DR5w12 15.3 9.6 3.4 2.2

A set of HBV-derived CTL epitopes that bind to multiple HLA supertypealleles has been identified (Table 7).

TABLE 7 HBV Vaccine HLA-A2, -A3 & -B7 CTL Epitopes HLA SEQ ID PrototypeAllele Immunogenicity Supertype Epitope Sequence NO: Conservation (%)¹Binding (IC₅₀ nM)² XRN³ Human⁴ Mice⁵ A2 core 18 FLPSDFFPSV 9 45 3.55 + + A2 env 183 FLLTRILTI 10 80 9.8 4 + + A2 env 335 WLSLLVPFV 11 1005.4 4 + + A2 env 338 LLVPFVQWFV 12 95 5.7 5 A2 env 378 LLPIFFCLWV 13 100158.9 1 A2 pol 455 GLSRYVARL 14 55 55.9 3 + + A2 pol 538 YMDDVVLGV 15 906.4 5 + + A2 pol 773 ILRGTSFVYV 16 A2 pol 562 FLLSLGIHL 17 95 7.8 3 + +A2 pol 642 ALMPLYACI 18 95 12.9 4 A2 env 338 GLSPTVWLSV 19 A3 core 141STLPETTVVRR 20 95  735/4.5* 4 + + A3 pol 149 HTLWKAGILYK 21 10015.4/15.6 5 + + A3 pol 150 TLWKAGILYK 22 100 2.1/33  2 A3 pol 388LVVDFSQFSR 23 100 6875/17   3 + + A3 pol 47 NVSIPWTHK 24 100 174/1173 + + A3 pol 531 SAICSVVRR 25 95 2189/29   3 + + A3/A1 pol 629 KVGNFTGLY26 95  58/365 2 A3 pol 665 QAFTFSPTYK 27 95 249/8   3 + + B7 core 19LPSDFFPSV 28 45 3026.8 4 + − B7 env 313 IPIPSSWAF 29 100 42.3 4 + + B7pol 354 TPARVTGGVF 30 90 13.2 2 + − B7 pol 429 HPAAMPHLL 31 100 56.6 4− + B7 pol 640 YPALMPLYA 32 B7 pol 541 FPHCLAFSY 33 B7 pol 530FPHCLAFSYM 34 95 58.5 5 − + B7 pol 640 YPALMPLY 35 B7 pol 640YPALMPLYACI 36 95 1393.4 3 − + ¹Sequence identity in 20 strains of HBVincluding adr, adw, ayr, and ayw isolates. ²Prototype alleles for therespective supertypes are A2: A*0201, A3: A*0301/A*1101, B7: B*0702.³Number of supertype alleles bound ≦500 nM. ⁴Recall CTL responses inpatients with chronic or active HBV infection. ⁵CTL responses induced inHLA-transgenic mice after immunization with a peptide emulsified in IFA.*Binding to HLA-A*0301 and -A*1101 respectively.

Six each of the HLA-A2, -A3 and -B7 supertype epitopes were selected foruse in vaccine development. The cutoff for binding affinity consideredwas 500 nM, since this level of binding affinity correlates with CTLimmunogenicity and antigenicity (Sette, A. et al., J. Immunol.153:5586-5592 (1994)). All of these epitopes are conserved in the mostprevalent HBV strains. The core 18 epitope is conserved in a relativelymodest 45% of the HBV sequences examined but the majority of thesequences that do not contain this particular epitope encode a variantwhich contains a conserved substitution (isoleucine for leucine) at theC-terminus of the epitope. All but one of the 18 selected epitopes bindat least three of the five the most common members of a given supertype.These epitopes were derived from the env, pol and core antigens,consistent with our goal to generate immune responses directed againstmultiple viral antigens, thus mimicking what the natural clearance ofHBV.

Human immune system recognition of these epitopes was demonstrated usingrecall CTL assays and PBL from individuals with either acute or chronicinfection (Bertoni, R., J. et al., J. Clin. Invest. 100:503-513 (1997)).Immune recognition of these epitopes by PBL demonstrates that theepitopes were produced in the course of natural HBV infection and thatthe appropriate TCR are present in the human repertoire. With theexception of three HLA-B7-restricted epitopes, the entire set of vaccineepitopes were recognized by CD8+ T-lymphocytes obtained from HBVpatients (Table 7).

The HLA-A2, -A3 and -B7 epitopes were also tested for immunogenicityusing HLA-transgenic animals. Following immunization with syntheticpeptides emulsified IFA, CTL responses were measured using an in situIFN-γ ELISA assay (Vitiello, A. et al., J. Clin. Invest. 95:341-349(1995)). Data obtained in this assay was converted to secretory units(SU) for evaluation (McKinney, D. M. et al., J. Immunol. Methods.237:105-117 (2000)). A SU is the number of cells that secrete 100 pg ofIFN-γ in response to a particular peptide, corrected for the backgroundamount of IFN-γ produced in the absence of peptide. The data shown inthe last column of Table 7 summarizes the findings of these experiments.The fact that most of these epitopes are immunogenic in HLA-transgenicmice is of relevance, as it offers a means of evaluating the potency ofmulti-epitope vaccines using a small animal model.

In conclusion, a set of epitopes suitable for inclusion in anepitope-based vaccine and restricted by three common HLA class Isupertypes can be untilized for vaccine development.

CTL Epitopes from the HLA-A1 and -A24 Supertypes

Epitopes binding to multiple members of the HLA-A1 and -A24 supertypeswere identified for the purpose of treating patients afforded by thevaccine and to increase the multiplicity of determinants contained inour epitope package (Table 8).

TABLE 8 HBV Vaccine HLA-A1 & -A24 CTL Epitopes HLA SEQ ID ConservationPrototype Allele Supertype Epitope Sequence NO: (%)¹ Binding (IC₅₀ nM)²XRN³ A1 env 359 WMMWYWGPSLY 37 85 16.3 3 A1 core 419 DLLDTASALY 38 752.3 3 A1 core 137 LTFGRETVLEY 39 75 80.0 3 A1 pol 149 HTLWKAGILY 40 100381.0 3 A1 pol 166 ASFCGSPY 41 100 247.0 3 A1 pol 415 LSLDVSAAFY 42 956.0 3 A1 pol 580 YSLNFMGY 43 85 382.0 3 A1 env 249 ILLLCLIFLL 44 100192.0 1 A24 env 236 RWMCLRRFII 45 95 11.0 3 A24 pol 392 SWPKFAVPNL 46 952.1 2 A24 env 332 RFSWLSLLVPF 47 100 12.0 2 A24 env 332 RFSWLSLLVPF 47100 12.0 2 A24 core 101 LWFHISCLTF 48 85 6.7 3 A24 core 117 EYLVSFGVW 4990 16.0 2 A24 pol 167 SFCGSPYSW 50 100 146.0 3 A24 pol 529 AFPHCLAF 5195 78.0 3 A24 pol 639 GYPALMPLY 52 95 280.0 2 A24 pol 745 KYTSFPWLL 5385 1.0 3 ¹Sequence identity in 20 strains of HBV including adr, adw,ayr, and ayw isolates. ²Prototype alleles for the respective supertypesare A1: A*0101, A24: A*2402. ³Number of supertype alleles bound ≦500 nM.

Of the over one hundred motif-positive peptides identified, 24 peptideswere selected for further study based on their binding characteristicsto purified HLA-A1 or -A24, and related supertype molecules and six eachrestricted to HLA-A1 or HLA-A24 were selected as vaccines; three relatedalleles were used to define the HLA-A1 and -A24 supertype families. Ofthese epitopes, core 117 and pol 745 were independently reported asbeing recognized by CTL from HBV-infected individuals (Sobao, Y. et al.,J. Hepatol. 34:922-929 (2001)).

To provide additional evidence showing that the selected epitopes couldbe recognized by human CD8+ T-lymphocytes, we induced primary CTLresponses using PBL obtained from non-infected normal donors. Thisin-vitro primary CTL induction assay utilizes PBL obtained byleukapheresis from HLA-A1 or -A24 positive, male and female donors. ThePBL were used as the source of dendritic cells (DC), antigen-presentingcells and CD8+ T-lymphocytes (Keogh, E. et al., J. Immunol. 167:787-796(2001)). To induce the expansion of precursor CTL to mature cells,purified CD8+ cells were co-cultured with cytokine-generated,peptide-pulsed DC in the presence of 10 ng/ml of recombinant human IL-7.This culture step induced the activation and initial maturation ofprecursor CTL, but restimulation and extended culture was needed toexpand their numbers for testing. The restimulation was done on days 7and 14 using adherent monocytes pulsed with peptide. Seven days afterthe second restimulation, the cultures were assayed for CTL activity,using either the in situ ELISA or the ELISPOT assays. A culture wasconsidered positive if the measured response is at least twice thebackground level of expression, determined using an irrelevant peptide,and ≧50 pg/well. A positive response demonstrates the presence of theappropriate TCR in humans.

A compilation of the data obtained using the HLA-A1 and -A24 epitopes isprovided in Table 9.

TABLE 9 Primary Immunogenicity of HLA-A1 & -A24 HBV CTL Epitopes DonorsHLA SEQ ID +/Total +Wells/Total Avg. Net IFN-γ Supertype EpitopeSequence NO: Tested¹ Tested² SI³ (pg/well)⁴ A1 env 359 WMMWYWGPSLY 371/4 1/192 23.0 175 A1 core 419 DLLDTASALY 38 1/3 3/144 29.0 67 A1 core137 LTFGRETVLEY 39 3/3 3/144 27.3 100 A1 pol 166 ASFCGSPY 41 2/4 3/19241.2 60 A1 pol 415 LSLDVSAAFY 42 1/4 1/192 57.0 56 A1 env 249 ILLLCLIFLL44 3/3 7/144 21.0 93 A24 env 236 RWMCLRRFIIF 45 0/2 0/96  A24 env 332RFSWLSLLVPF 47 1/2 1/96  2.5 186 A24 core 101 LWFHISCLTF 48 1/2 2/96 2.8 248 A24 core 117 EYLVSFGVWI 49 1/2 1/96  2.3 158 A24 pol 392SWPKFAVPNL 46 0/3 0/144 A24 pol 745 KYTSFPWLL 53 2/2 10/96   108.0 144¹Number of donors with positive CTL response of total number of donorstested. ²Number of cultures with positive CTL response of total culturestested. ³Average stimulation index of CTL responses calculated as:IFN-γ secretion with peptide/IFN-γ secretion without peptide. ⁴NetIFN-γ production adjusted for control irrelevant peptide.

The data is expressed as the number of positive wells out of the totalwells tested, the average stimulation index of the positive cultures andthe net IFN-γ release of the positive cultures. Significant CTLresponses were induced for all of the HLA-A1 restricted epitopes and for4/6 of the HLA-A24 epitopes included in the current HBV vaccine studies.

HLA-A1 and -A24 transgenic mice are not currently available. However, asignificant degree of similarity exists between the binding motifs ofHLA-A24 epitopes and the murine class I K^(d). We therefore tested fourof the vaccine HLA-A24 epitopes for their capacity to bind purifiedK^(d) molecules in vitro and assessed immunogenicity. We found that oneof these epitopes was immunogenic in the H2^(bxd) mice (Table 10). Twoother epitopes were not tested for binding but proved to be immunogenicwhen tested in H2^(bxd) mice following immunization with peptide/IFAemulsions. IFN-γ responses after in vitro restimulation ranged from158.7 to 339.6 SU. This level of activity was similar to the levelsobserved with a known control K^(d)-restricted epitope (Romero, et al.,Nature 341: 323. (1989)). Thus, the H2^(bxd) can be used in the absenceof HLA-A24 transgenic mice.

TABLE 10 Cross Reactivity of HLA-A24 Epitopes with K^(d) SEQ ID K^(d)Binding Murine Epitope Sequence NO: Conservation (%)¹ (IC₅₀ nM)Immunogenicity² (SU) env 236 RWMCLRRFII 45 95 NT 339.6 pol 392SWPKFAVPNL 46 95 NT 261.0 env 332 RFSWLSLLVPF 47 100 env 332 RFSWLSLLVPF47 100 — 0.0 core 101 LWFHISCLTF 48 85 — 0.0 core 117 EYLVSFGVW 49 90 —0.0 pol 167 SFCGSPYSW 50 100 pol 529 AFPHCLAF 51 95 pol 639 GYPALMPLY 5295 pol 745 KYTSFPWLL 53 85 77.5 158.7 CS 252³ SYIPSAEKI 54 NA 9.2 49.2¹Sequence identity in 20 strains of HBV including adr, adw, ayr, and aywisolates. ²CTL response, measured in an in situ ELISA assay, (McKinneyet al. 2000) after peptide/IFA immunization of H2^(bxd) mice. ³ControlA24 epitope (Romero et al. 1991). NT: not tested NA: not applicableC. Projected Population Coverage of Vaccines Composed of CTL Epitopes

The population coverage of vaccines composed of the selected epitopeswas determined based on the phenotypic frequencies of HLA types definedby the HLA workshop and on the binding characteristics of the epitopes(Gjerston, D. W. and Terasaki, P. I. HLA. American society forHistocompatibility and Immunogenetics. Lenexa, Kans. (1998)).

The most common HLA molecules contained within each of the five selectedHLA class I supertypes and their distribution in common ethnic groups isshown in Table 6A. The calculated phenotypic frequency of individualswith HLA types capable of binding the indicated number of classI-restricted epitopes and the cumulative frequency of individualspredicted to be genetically capable of responding immunologically to theselected epitopes is shown in FIGS. 25A-25B. An average of 11.1epitope-HLA combinations could be recognized in an idealized compositepopulation with average HLA frequencies (FIG. 25A). Analyses ofprojected population coverage in the major ethnic groups demonstrated noappreciable ethnic bias (FIG. 25B).

Selection of HTL Epitopes

HLA-DR types can be grouped into two major supertypes based onepitope-peptide binding, defined as the HLA-DR-1, 4, 7 and -DR3supertypes (Wilson, C. C. et al., J. Virol. 75:4195-4207 (2001); Doolan,D. L. et al., J Immunol. 165:1123-1137 (2000); Southwood, S. et al., J.Immunol. 160:3363-3373 (1998)). A set of HBV-derived, HLA-DR supertypeepitopes was identified using a process similar to that used to identifythe CTL epitopes and 16 were selected for further study based on bindingcharacteristics (Table 11).

TABLE 11 HBV Vaccine HTL Epitopes HLA (SEQ HLA-DR binding capacity (IC₅₀nM) Super- ID Conserva- DRB1 DRB1 DRB1 DRB1 DRB1 DRB1 DRB1 DRB1 DRB1DRB1 DRB1 DRB5 DRB3 DRB4 # DR type Epitope Sequence NO:) tion (%)¹ *0101*1501 *0301 *0401 *0405 *1101 *1201 *1302 *0701 *0802 *0901 *0101 *0101*0101 bound² DR pol 412 LQSLTNLLSSNLSWL (55) 90 2.0 21 — 10.0 47 303 397143 173 598 791 1067 1837 4179 10 po1 664 KQAFTFSPTYKAFLC (56) 60 10 41— 88 181 82 — 190 90 416 142 144 4848 322 11 env 180 AGFFLLTRILTIPQS(57) 80 1 217 — 9 258 6 4229 9 8 189 56 1158 4374 696 10 pol 774GTSFVYVPSALNPAD (58) 80 15 748 — 119 94 443 — — 94 818 220 400 — — 9core 120 VSFGVWIRTPPAYRPPNAPI (59) 90 27 43 — 58 220 11 817 565 78 761773 7 6454 395 8 pol 145 RHYLHTLWKAGILYK (60) 100 17 4.0 — 2271 1499 42149 766 61 36 133 35 — 782 10 env 339 LVPFVQWFVGLSPTV (61) 95 408 14 —315 28 54 452 2330 2744 60 31 1516 1661 22 9 pol 501 LHLYSHPIILGFRKI(62) 80 248 558 — 77 244 492 9462 — — 800 1551 560 — 102 8 pol 523PFLLAQFTSAICSVV (63) 95 27 359 — 560 246 1749 — 59 328 940 1373 4764 —1347 7 pol 618 KQCFRKLPVNRPIDW (64) 45 3.0 4370 — 40 34 1617 — 821 62872 5175 1246 — 3060 6 pol 767 AANWILRGTSFVYVP (65) 70 55 386 — 966 16341520 802 143 44 214 299 3276 — 6553 8 core 50 PHHTALRQAILCWGELMTLA (66)90 810 8.0 — 326 — 458 — — 676 210 952 124 575 48 7 DR3 pol 694LCQVFADATPTGWGL (67) 95 7470 5009 67 490 1203 — — 2022 — — — — 1808 10442 pol 385 ESRLVVDFSQFSRGN (68) 45 7372 1368 36 208 251 — — 946 — — — —2525 8711 3 pol 96 VGPLTVNEKRRLKLI (69) 60 8415 4153 43 3916 1908 6666 —4461 — 5354 — 4330 — 8121 1 pol 420 SSNLSWLSLDVSAAF (70) 85 38 3089 62168 17 4923 1859 36 5063 1065 7126 — 5 7 4 ¹Sequence identity in 20strains of HBV including adr, adw, ayr, and ayw isolates. ²Number of DRalleles bound with IC₅₀ ≦ 1000 nM.

The immunogenicity of the vaccine HTL epitopes was evaluated in both HBVpatients and mice (Table 12).

TABLE 12 Immunogenicity of HBV Vaccine HTL Epitopes HLA ImmunogenicityHLA overlaps Core Freq SEQ ID HBV H2^(bxd) Supertype Alt pep with AgEpitope Sequence (X/20) NO: patients¹ mice² LQSLTNLLSS DR 1186.13 HBVpol 412 NLSWL 18 55 + + KQAFTFSPT HBV pol 664 YKAFLC 19 56 + +AGFFLLTRIL 830.01 1280.08 HBV env 180 TIPQS 16 57 + + GTSFVYVPS HBV pol774 ALNPAD 18 58 + + VSFGVWIRT PPAYRPPNA 1186.25 HBV core 120 PI `59 + + RHYLHTLW HBV pol 145 KAGILYK 20 60 + + LVPFVQWFV HBV env 339GLSPTV 19 61 + − LHLYSHPIIL HBV pol 501 GFRKI 16 62 + + PFLLAQFTSA1186.19 1186.26 HBV pol 523 ICSVV 19 63 + + KQCFRKLPV HBV pol 618 NRPIDW16 64 + − AANWILRGT HBV pol 767 SFVYVP 16 65 + − PHHTALRQA ILCWGELMTF039.01 HBV core 50 LA 66 + − LCQVFADAT DR3 35.0100 HBV pol 694 PTGWGL19 67 + + ESRLVVDFS HBV pol 385 QFSRGN 20 68 + + VGPLTVNEK HBV pol 96RRLKLI 69 − + SSNLSWLSL 1186.18 HBV pol 420 DVSAAF 20 70 − + ¹Recall CTLresponses in patients with chronic or active HBV infection ²HTLresponses induced in H2^(bxd) mice after immunization with a peptide/CFAemulsion.

With the exception of two HLA-DR3 epitopes, all epitopes are recognizedin HBV-infected humans. The immunogenicity of the HTL epitopes was alsocharacterized using H2^(bxd) mice. Epitope-peptide binding preferencesare similar for HLA-DR1 and IA^(b) allowing for comparison testing(Wall, K. A. et al., J. Immunol. 152:4526-4536 (1994)) in non-transgenicmice. Twelve of the HTL epitopes were immunogenic in these mice, asjudged by fresh ELISPOT assays performed 11-14 days after immunizationwith 25 μg of purified, synthetic peptides (Table 12).

In conclusion, these data identify a set of HTL epitopes suitable forinclusion in an HBV vaccine construct.

Projected Population Coverage at the Level of HTL Epitopes

The selected HTL epitopes were derived from the core, pol and envantigens, thus offering the opportunity of generating multi-specificresponses in immunized individuals. These epitopes also provide a highlevel of predicted population coverage across the most common ethnicgroups. Table 6B summarizes the HLA types included in the analysis andtheir distribution in common ethnic groups. The calculated phenotypicfrequency of individuals with HLA types capable of binding the indicatednumber of class II-restricted epitopes and the cumulative frequency ofindividuals predicted to be genetically capable of respondingimmunologically to the selected epitopes is shown in FIGS. 26A-26B. Wepredict that an average of 17.2 epitope-HLA combinations could berecognized in an idealized population composed of averaging HLAfrequencies observed in major ethnic groups (FIG. 26A). The averagenumber of epitope-combinations potentially recognized is higher than 16(the total number of epitopes) because the heterozygosity and of thehighly degenerate binding capacity of the epitopes. Analysis of themajor ethnic groups demonstrated very broad population coverage (FIG.26B).

3. Minigene Construct Design

Background

The focus of our research is the development of vaccine constructscomposed of multiple epitopes. Studies from a number of differentlaboratories demonstrated that multi-epitope constructs can beconfigured by stringing epitopes one after the other in a “string ofbeads” manner. However, the immunogenicity of individual CTL epitopes inconstructs of this type is often highly variable. Variation can beattributed to the differential efficiency of the cellular processingthat generates epitopes. We found that the use of appropriate amino acidspacers to ensure efficient proteosomal cleavage results in balancedepitope processing and immunogenicity. (Velders, M. P. et al., J.Immunol. 166:5366-5373 (2001); Livingston, B. D. et al., Vaccine.19:4652-4660 (2001)).

The possibility of creating artificial epitopes, referred to as“junctional epitopes,” has been considered. Junctional epitopes maydominate or redirect responses in an inappropriate manner and/or may behomologous to human (self) sequences and thereby induce anti-selfresponses. A computer program has been designed that, for each epitopepair, selects the spacer composition that optimizes proteosomal cleavageand minimizes the occurrence of epitope motifs through the addition ofadditional amino acids as spacers. Thus, our epigene construct designsoftware evaluates different epitope arrangements and selects those withoptimal predicted proteosomal cleavage and minimal occurrence rate ofjunctional motifs.

Optimization of HLA-DR binding epitopes for proteosomal cleavage is notrelevant, although avoiding junctional epitopes remains a primary designconsideration. Since the motifs recognized by HLA class II molecules aremore broadly defined, we designed a strategy based on the use of auniversal spacer consisting of GPGPG (SEQ ID NO:2); (Livingston et al.J. Immunol. 168:5499-5509 (2002)). This spacer has the capacity ofdisrupting binding to most, if not all, of the most common HLA-DR typessince it is poorly compatible with the majority of human and murineclass II binding motifs (Livingston, B. et al., J. Immunol.168:5499-5506 (2002)).

The N-terminus of the epigene CTL constructs includes the sequenceMGMQVQIQSLFLLLLWVPGSRG (e.g., amino acids 1-22 of SEQ ID NO:72). This isa consensus sequence based on the Ig kappa secretory signal, andincluded to assist in MHC Class I targeting of the polypeptide product.

Another important element of the vaccine design strategy is theinclusion of a universal HTL epitope (Alexander, J. et al., Immunity1:751-761 (1994)). This non-natural epitope was designed to bind to themost common HLA molecules with high affinity and for optimalimmunogenicity by maximizing TCR contact residues. This HTL epitope caninduce HTL responses to support the induction and augmentation of CTLresponses (Alexander, J. et al., Immunity 1:751-761 (1994); Alexander,J. et al., Immunol. Res. 18:79-92 (1998)). Utilization of anon-HBV-derived HTL epitope might offer unique advantages in terms ofsupport for CTL induction in the chronic setting because HBV-specificHTL responses may, in part, be impaired by the tolerance associated withchronic infection. In fact, it has been demonstrated that this HTLepitope allows the immune system to overcome HBV-specific T celltolerance in transgenic mice expressing HBV antigens (Livingston, B. D.et al., Hum. Immunol. 60:1013-1017 (1999); Livingston, B. D. et al., J.Immunol. 162:3088-3095 (1999); Alexander, J. et al., Immunol. Res.18:79-92 (1998); Sette, A. D. et al., J. Immunol. 166:1389-1397 (2001)).This HTL epitope is also included in HTL vaccine constructs because itenhances responses induced by other HTL epitopes. This “help for thehelpers” concept is consistent with recently published observation inthe CD40 system, which suggests that dendritic cell licensing, definedas HTL-induced upregulation of accessory molecules on dendritic cells,can also apply to HTL responses (Gerloni, M. S. et al., Proc. Natl.Acad. Sci. USA. 97:13269-13274 (2000); van Mierlo, G. J. et al., Proc.Natl. Acad. Sci. USA. 99:5561-5566 (2002)).

B. Design of a Minigene Construct Encoding HBV-Derived CTL Epitopes

A first prototype vaccine construct, HBV1, included 17 HLA-A2, -A3 and-B7 epitopes and lacked amino acid spacers. This construct was modified(HBV2) to incorporate appropriate spacers and increase theimmunogenicity of a number of the component epitopes. HBV2 induced CTLresponses to a wide spectrum of epitopes that were in general comparableto those induced by immunization with peptides emulsified in IFA (datanot shown). This type of control allows one to estimate the activitydetectable for each particular epitope in the absence of any processingconstraint, and thus allows standardization of factors such asavailability and size of an epitope-specific TCR repertoire in thevarious strains of mice utilized for preclinical evaluations.

A number of new epigene constructs were designed to include HLA-A1 and-A24 epitopes to provide greater population coverage. Four epigeneconstructs incorporating 21 and 30 CTL epitopes were constructed andtested for immunogenicity, focusing on the HLA-A2 epitopes. All fourconstructs induced broad, potent CTL responses (data not shown). As the30 epitope constructs should provide a greater redundancy of epitopecoverage in the prospective patient population, additional studiescentered on these larger epigene constructs. One particular 30 epitopeconstruct, HBV30C, induced strong CTL responses to both the HLA-A3 and-A24 epitopes (the latter measured using H2^(bxd) mice). Although two ofthe HLA-A2 epitopes, core 18 and env 183, were poorly immunogenic inthis construct, further spacer optimization restored the immunogenicityof these epitopes. A schematic and the amino acid sequence of the CTLvaccine HBV30K are shown in FIG. 27A and Table 13. An example of apolynucleotide sequence encoding HBV30K is shown in Table 13.

The immunogenicity of HBV30K in HLA-A2 and -A11 transgenic mice is shownin FIG. 27B. As a means of comparison, the CTL activity inducedfollowing immunization with the potent HBV2 prototype construct as wellas peptide/IFA is also shown. Overall, HBV30K elicited CTL responses asvigorous as HBV2. In fact, HBV30K induced CTL responses against all thecomponent epitopes that are immunogenic in the HLA transgenic animalsand typically these CTL responses were comparable to the responsesinduced following peptide immunization. This data lead to the selectionof HBV30K as the lead CTL vaccine.

TABLE 13 HBV30K construct HBV30K Polynucleotide SEQ ID NO: 71

CACGGGGCTTCTTGCTTAGCTTGGGCATCCACCTAAATGCTGCTGCAAAATACACATCTTTTCCTTGGCTCCTTAATGCCGCCGCTAGGTTTTCATGGCTGAGTCTGCTAGTACCTTTCAATGCGGCTTTCCCACATTGCCTAGCTTTTAGCTATATGAAAGCTGCTTTAGTCGTGGACTTTTCACAGTTTAGCAGAGGAGCAATCCTGCTGCTATGTCTGATATTCCTTCTAAACGCAGCAGCCCACACACTCTGGAAAGCTGGTATCCTTTACAAGAAAGCCTGGATCATGTGGTATTGGGGACCCAGCCTCTACAAAGCATACCCTGCCCTGATGCCACTATACGCATGCATTGGCGCGGCAGCCTGGTTATCCCTTTTAGTACCGTTTGTCAACGCCGCAGCGGGATTTCTATTAACCAGAATCCTGACGATTAATGCTGCCGCCATTCCGATCCCAAGTTCCTGGGCATTCAAAGCAGCCGCGGAGTATCTGGTTTCATTTGGCGTATGGAACCTGCCAAGCGACTTCTTTCCTTCTGTTAAGGCCGCTGCTTTCCTCCCCTCCGATTTCTTTCCATCGGTGAAAGCCGCTGCCGACCTCCTTGATACCGCGAGCGCTCTGTACAACTCGTGGCCAAAATTCGCAGTTCCAAACCTAAAAGCCGCCGCCAGTGCCATTTGTTCCGTGGTAAGGAGAAAATTATCACTCGACGTGTCCGCAGCATTTTATAACGCTGCTGCAAAGTTTGTCGCAGCATGGACATTGAAGGCTGCAGCGAAAGCAGCAAATGTATCAATACCCTGGACCCACAAGGGTGCAGCCGGGCTGTCTAGGTATGTGGCGAGGCTAAACGCCGCCGCCTCAACACTGCCTGAGACTACTGTCGTGAGACGCAAACACCCTGCCGCAATGCCCCACCTGCTGAAAGCAGCCGCACGATGGATGTGCCTCAGAAGATTCATAATAAACGCTTCTTTCTGTGGGTCACCCTACAAAGCCGCTTACATGGACGATGTGGTCCTCGGAGTGAATGCCCTCTGGTTCCATATCAGCTGCCTGACATTCAAGGCAGCCGCCACCCCCGCTCGTGTGACAGGAGGTGTCTTCAAAGCCGCGGCACTGACTTTCGGTCGGGAAACTGTATTGGAATATAAGCA

HBV30K Polypeptide SEQ ID NO: 72

AAFPHCLAFSYMKAALVVDFSQFSRGAILLLCLIFLLNAAAHTLWKAGILYKKAWMMWYWGPSLYKAYPALMPLYACIGAAAWLSLLVPFVNAAAGFLLTRILTINAAAIPIPSSWAFKAAAEYLVSFGVWNLPSDFFPSVKAAAFLPSSFFPSVKAAADLLDTASALYNSWPKFAVPNLKAAASAICSVVRRKLSLDVSAAFYNAAAKFVAAWTLKAAAKAANVSIPWTHKGAAGLSRYVARLNAAASTLPETTVVRRKHPAAMPHLLKAAARWMCLRRFIINASFCGSPYKAAYMDDVVLGVNALWFHISCLTFKAAATPARVTGGVFKAAALTFGRETVLEYKQAFTFSPTY

C. Design of a Minigene Construct Encoding HBV Derived HTL Epitopes

A single epigene construct encoding the 16 HTL epitopes was designedincorporating the GPGPG (SEQ ID NO:2) universal spacer. A schematic andthe amino acid sequence of this HBV HTL construct are shown in FIG. 28Aand Table 14. An example of a polynucleotide sequence encoding the HBVHTL construct is shown in Table 14. This construct was tested forimmunogenicity in H2^(bxd) mice (FIG. 28B), measuring IFN-γ productionby CD4+ T-lymphocytes using an ELISPOT assay. Responses were as vigorousas those induced by a peptides emulsified in CFA for 50% of the epitopes( 6/12) tested. Of the remaining six epitopes, and only two epitopesfailed to induce a response following immunization with the HTL vaccineconstruct.

TABLE 14 HBV HTL construct HTL Polynucleotide SEQ ID NO: 73

GGCCTGTGCCAG GTCTTCGCCGACGCAACTCCCACAGGGTCCGGGCTGGGGCCAGGACCAGGCAGGCACTACCTGCATACTCTGTGGAAGGCAGGAATCCTCTATAAAGGGCCCGGCCCAGGCCCTCACCACACCGCCCTGAGGCAGGCCATCCTGTGCTGGGGGGAGCTCATGACCCTGGCCGGACCTGGACCCGGGGACAGCACACTGGTGGTGGATTTCAGCCAATTCAGCAGAGGAAACGGACCCGGCCCTGGGCCTTTTCTGCTGGCTCAGTTTACATCTGCTATTTGTTCTGTGGTCGGCCCCGGGCCCGGACTCGTGCCTTTCGTGCAGTGGTTCGTGGGACTGTCCCCTACAGTCGGGCCCGGCCCAGGGCTGCATCTGTACTCCCACCCAATCATCCTCGGCTTCCGCAAGATTGGACCCGGCCCAGGCTCCAGCAATCTCTCCTGGCTCTCTCTGGACGTGTCTGCCGCCTTTGGCCCTGGACCAGGCCTGCAAAGCCTGACTAATCTGCTCAGCAGCAACCTGTCCTGGCTGGGACCTGGCCCAGGGGCTGGCTTCTTTCTGCTCACCCGGATTCTCACAATTCCCCAGTCCGGACCAGGACCAGGAGTCAGTTTCGGGGTGTGGATCAGGACCCCTCCTGCTTATAGACCACCCAATGCTCCAATCGGCCCCGGCCCTGGCGTCGGGCCACTGACCGTGAATGAGAAGCGCCGGCTGAAGCTGATCGGCCCTGGCCCTGGCAAGCAGTGCTTTCGCAAACTGCCCGTGAACAGACCTATTGATTGGGGCCCCGGCCCTGGAGCAGCCAACTGGATTCTCAGGGGAACAAGCTTCGTCTACGTGCCCGGGCCCGGACCAGGGAAGCAGGCTTTTACCTTCTCTCCCACTTACAAGGCCTTCCTCTGTGGGCCAGGCCCCGGCGCCAAGTTTGTGGCAGCATGGACCCTCAA

HTL Polypeptide SEQ ID NO: 74

GPHHTALRQAILCQGELMTLAGPGPGESRLVVDFSQFSRGNGPGPGPFLLAQFTSAICSVVGPGPGLVPFVQWFVGLSPTVGPGPGLHLYSHPIILGFRKIGPGPGSSNLSWLSLDVSAAFGPGPGLQSLTNLLSSNLSWLGPGPGAGFFLLTRILTIPQSGPGPGVSFGCWIRTRPAYRPPNAPIGPGPGVGPLTVNEKRRLKLIGPGPGKQCFRKLPVNRPIDWGPGPGAANWILRGTSFVYVPGPGPGKQ

Effective Minimization of Junctional Epitope Content

After defining epigene constructs for the CTL and HTL vaccineconstructs, we proceeded with a more in-depth characterization. First,we verified that the computer-based epigene construct design efforteffectively minimized the presence of junctional epitopes. Thejunctional epitope content of the CTL and HTL components was determinedusing a motif scan and compared to two sets of random assortments of thesame CTL and HTL epitopes. The results are shown in Table 15.

TABLE 15 Example of minimization of junctional epitopes in vaccineconstructs SEQ ID Junctional Construct Protocol NO: CTL Motifs⁴ HBV30K¹minimized 1 HBV30R1 random² 84 HBV30R2 random 99 HBV HTL¹ GPGPG 2 12 HBVHTL NS1 no spacer³ 42 HBV HTL NS2 no spacer 37 ¹Vaccine CTL and HTLepigene constructs. ²Random arrangement of CTL epitopes optimized forprocessing. ³HTL epigene constructs without spacers. ⁴Number ofjunctional epitopes bearing HLA-A1, -A2, -A3, -A24 or B7 motifs.

The number of junctional epitopes present in the optimally designed CTLepitope vaccine is approximately 100-fold lower, compared to randomarrangements. While the HTL component was not specifically minimized forthe presence of junctional CTL epitopes, the use of the GPGPG spacer(SEQ ID NO:2), to eliminate HTL functional epitopes within the string ofHBV-specific HTL epitopes, did reduce the presence of junctional CTLepitopes by approximately 4-fold. Junctional HTL epitopes were notconsidered in the analysis of the CTL epitope string as the presence ofsuch epitopes in the CTL epigene construct should only serve tostimulate non-specific help much in the same way as the universal HTLepitope mentioned above (Alexander, J. et al., Immunity 1:751-761(1994)).

BLAST searches were performed to evaluate the potential for homology ofjunctional regions in the HBV CTL and HTL epigene constructs. As inputsequences, we considered each of the 47 sequences comprised of fourC-terminal residues of an epitope, the spacer sequence itself, ifpresent, and the four N-terminal residues of the following epitope. Forthe BLAST search parameters, we used the search option “Short nearlyexact matches.” To run the search with the least stringent criteria, weused the default settings present on the web page; expect value at20,000 and word size set at 2. The organism field was restricted to Homosapiens. Table 16 lists the results.

No junctional region was 100% homologous to any human sequence. Thehighest homology was 78% and the least was 54% (mean 63±7.4). For thesake of comparison, an identical homology search was run on a randomsample of seven CTL and four HTL HBV epitopes (Table 17).

The best homology detected was 67% and the least was 30% (mean 54±13.

TABLE 16 Results of human homology search based on epigene constructjunctional motifs Junctional SEQ ID % Region Source NO Sequence HomologyAccession No. 1 pol 562-NAAA-pol 75 GIHLNAAAKYTS — 745 Unknown protein76 GIHLN*AA**** 58 AAH14187 for MGC:20975 2 pol 745-NAAA-env 77PWLLNAAARFSW — 332 sulfonylurea 78 PWLLNA****** 50 AAC36724.1 receptor 13 env 332-NAA-pol 530 79 LVPFNAAFPHC — KIAA1219 protein 80 ***F+AAF*HC55 BAA86533.2 4 pol 530-KAA-pol 388 81 FSYMKAALVVD — Hypothetical 82FSYMKAA**** 63 XP_073807.1 protein 5 pol 388-GA-env 249 83 QFSRGAILLL —Hypothetical 84 QFS*GAIL** 70 XP_066589.1 protein Hypothetical 85*FSR*AILL* 70 NP_071768.2 protein FLJ22313 6 env 249-NAAA-pol 86IFLLNAAAHTLW — 149 Hypothetical 87 **LLNA**H*LW 58 XP_060325.1 protein 7pol 149-KA-env 359 88 ILYKKAWMMW — Nebulin 89 ILYK*AW*** 60 AAB02622.1Hypothetical 90 *****AWMMW 50 AAH21093 protein FLJ14753 8 pol 149-KA-pol640 91 PSLYKAYPAL — Intergrase interactor 92 *SLYK*YP+L 70 AAA81905.1 19 pol 640-GAA-env 335 93 YACIGAAWLSL — Steroid 18- 94 **C+*A*WLSL 55AAB34642.1 hydroxylase CGI-67 protein 95 YA*I*AAWL+L 73 AAD34062.1 10env 335-NAAA-env 96 VPFVNAAAFLLT — 183 Hypothetical 97 *PFVNA**FL** 58CAD38882.1 protein K1AA1742 protein 98 *PFVN*AA*LL* 67 BAB21833.2 11 env183-NAAA-env 99 ILTINAAAIPIP — 313 hRANKL2 100 *LTHINA**IP** 58AAC517.62.1 12 env 313-KAAA-core 101 SWAFKAAAEYLV — 117 AP-2 beta 102****KAAAEYL* 58 CAC04182.1 transcription factor 13 core 117-N-core 19103 FGVWNLPSD — Hypothetical 104 *GVWNL*SD 78 CAD38975.1 protein 14 core19-KAAA-core 105 FPSVKAAAFLPS — 18 Nascent 106 *PS*KAAAFL** 67XP_061543.1 polypeptide- associated complex 15 core 18-KAAA-core 107FPSVKAAADLLD — 419 Zinc finger protein 108 **SVKAA++LL* 58 XP_087479.464 16 core 419-N-pol 392 109 SALYNSWPK — Immunoglobulin 110 ***YN+WPK 55AAM46537.1 kappa VLJ region 17 pol 392-KAAA-pol 111 VPNLKAAASAIC — 531DNA poly. epsilon 112 **NLKAAAS*** 58 AAA15448.1 catalytic subunit 18pol 531-K-pol 415 113 VVRRKLSLD — Hypothetical 114 V+RRK+SLD 78XP_064183.1 protein 19 pol 415-NAA-padre 115 AAFYNAAAKFV — Potassiumvoltage- 116 *AFYN*A+KF* 64 AAH27932.1 gated channel 20 padre-KAA-pol 47117 KAAAKAANVSI — Laminin beta 118 KAA*KAAN+** 64 AC005048.1 precursor21 pol 47-GAA-pol 455 119 WTHKGAAGLSR — Hypothetical 120 WTHKG+*GL+R 73XP_117843.1 protein 22 pol 455-NAAA-core 121 VARLNAAASTLP — 141 Solutecarrier 122 VARL+AAA**** 58 NP_060237.1 family 39 (zinc transporter)Hypothetical 123 VA*L*AAA+TL* 67 XP_120525.1 protein 23 core 141-K-pol429 124 VVRRKHPAA — Hypothetical 125 VRRKHP*A* 78 XP_117855.1 protein 24pol 429-KAAA-env 126 PHLLKAAARWMC — 236 Hypothetical 127 **LL*AA*RW*C 58XP_105701.1 protein 25 env 236-N-pol 166 128 RFIINASFC — Hypothetical129 RFII+A*F* 67 XP_072766.5 protein 26 pol 166-KAA-pol 538 130GSPYKAAYMDD — Hypothetical 131 **PY**AYMD* 54 AAH01463 protein 27 pol538-NA-core 101 132 VLGVNALWFH — Hypothetical 133 **GV+ALWF* 60XP_118305.1 protein Hypothetical 134 VL*+*ALWFH 70 XP_059358.1 protein28 core 101-KAAA-pol 135 CLTFKAAATPAR — 354 KIAA 1853 protein 136**TFKA*ATP** 58 BAB47482.1 Alpha 1 type XIII 137 **T*KAAAT*AR 67NP_542994.1 collagen 29 pol 354-KAAA-core 138 GGVFKAAALTFG — 137 Unknown139 *GV**AA+LTFG 67 AE006639.1 30 core 137-K-pol 665 140 VLEYKQAFT —Hypothetical 141 VL+YKQ*F* 67 XP_101671.1 protein X-linked mental 142VL*YKQ*FT 78 CAA65075.1 retardation cand. gene 31 pol 665-GPGPG-pol 143PTYKGPGPGGTSF — 774 sialyltransferase 1 144 **YKGPGPG**** 54 CAA35111.1N2B-Titin isoform 145 **YK*PGP*GT*F 61 CAD12455.1 32 pol 774-GPGPG-pol146 NPADGPGPGLCQV — 694 golgi antigen 147 NPAD*PGPG**** 61 AAC06338.1 33pol 694-GPGPG-pol 148 GWGLGPGPGRHYL — 145 L-myc-1 proto- 149*WGLGPG*G**** 54 AAA59879.1 oncogene protein 34 pol 145-GPGPG-core 150ILYKGPGPGPHHT — 50 sialyltransferase 1 151 **YKGPGPG**** 54 CAA35111.135 core 50-GPGPG-pol 152 MTLAGPGPGESRL — 385 Hypothetical 153**LAGPGPG*SR* 69 XP_069591.1 protein Mitogen-activated 154 ****GPG*GESRL61 XP_027237.1 protein kinase 36 pol 385-GPGPG-pol 155 SRGNGPGPGPFLL —523 protein kinase C mu 156 **G+GPGP*PFL* 61 CAA53384.1 CD1-alpha-3 157SRG**PGPG**LL 69 AAA51935.1 antigen 37 pol 523-GPGPG-env 158CSVVGPGPGLVPF — 339 Inducible nitric 159 C*++GPG*G+VPF 61 AAL02120.1oxide synthase 38 env 339-GPGPG-pol 160 SPTVGPGPGLHLY — 501 Atrophin-1161 SPTVGPGP***** 61 S50832 39 pol 501-GPGPG-pol 162 FRKIGPGPGSSNL — 420Hypothetical protein 163 *RKI*PGPG**** 54 XP_069589.1 Hypotheticalprotein 164 *RKI**G*GSSN* 61 XP_169769.1 40 pol 420-GPGPG-pol 165SAAFGPGPGLQSL — 412 Epsin 2b protein 166 S*+FGPGPG++S+ 61 AAC78609.1 41pol 412-GPGPG-env 167 LSWLGPGPGAGFF — 180 Hypothetical protein 168LSWLGPG*G**** 61 XP_097563.1 Unnamed protein 169 ***LGPGP**GFF 61BAC05301.1 product 42 env 180-GPGPG-core 170 IPQSGPGPGVSFG — 120Transmembrane 171 **PQ+GPGPGV** 61 AAC64943.1 protein 43 core120-GPGPG-pol 172 NAPIGPGPGVGPL — 96 Unnamed protein 173 ****GPGPG*GPL61 BAC05043.1 product Neuregulin 2 174 *AP*GPGPG*GP* 69 AAF28851.1isoform 4 44 pol 96-GPGPG-pol 175 LKLIGPGPGKQCF — 618 Unknown protein176 LKL*GPGPG**** 61 AF318376.1 45 pol 618-GPGPG-pol 177 PIDWGPGPGAANW —767 Hypothetical protein 178 **DWGPGPG**** 54 XP_066062.1 46 pol767-GPGPG-pol 179 VYVPGPGPGKQAF — 664 TAF4 RNA 180 ***PGPGPGK*A* 61XP_036470.2 polymerase II 47 pol 664-GPGPG-padre 181 AFLCGPGPGAKFV —Polycystic kidney 182 **LGGP*PGA*** 54 AAC37576.1 disease 1 protein 1.Spacer groups with the 4 adjacent residues from neighboring epitopeswere utilizied as query sequences. 2. BLAST search parameters: Expect20000, Word size 2, Matrix PAM30, No. of alignments 250 3. Resultantsequence matches with the lowest E value are presented first. Nosequence identified had an E value <1. 4. If additional sequences wereidentified with greater homology, they are also presented. 5. Asterisks(*) denote non-matching residues; plus signs (+) denote residues ofsimilar chemical composition.

TABLE 17 Results of human immunology search using random epitope orderSEQ Junctional ID % Accession Region Source NO: Sequence Homology No. 4pol 530 183 FPHCLAFSYM — Endothelin receptor B delta 3 184 **HCLAFS** 60AF114165.1 8 pol 149 185 WMMWYWGPSLY — Hypothetical protein 186WMMW*W***** 45 NP_076417.1 FLJ12389 11 env 183 187 FLLTRILTI —Hypothetical protein DKFZ 188 *LLTR+LT* 67 AAH30825.1 13 core 117 189EYLVSFGVW — Glucose 6-phosphate 190 *YLV*FGV* 67 CAA75608.1 translocase15 core 18 191 FLPSDFFPSV — T-ceIl activation NFKB-like 192 FLP*DF+P**60 NP_116110.2 protein 18 pol 531 193 SAICSVVRR — Membrane-spanning 4-194 SAICS*V** 67 AF237905.1 domains (MS4A8B) protein 24 pol 429 195HPAAMPHLL — Hypothetical protein 196 H*AAMPH** 67 XP_120541.1 35 core 50197 PHHTALRQAILCWGE — LMTLA Cysteine dioxygenase 198 *********ILCWGE****30 BAA12873.1 * 36 pol 385 199 ESRLVVDFSQFSRGN — B/K protein 200****VVDF*+FSR** 47 NP_057608.1 38 env 339 201 LVPFVQWFVGLSPTV —Cytochrome B561 202 ***FVQW*VG*S*** 47 AAC50212.1 47 pol 664 203KQAFTFSPTYKAFLC — hypothetical protein 204 *Q*FTF*PT++A*** 47 AAH07800FLJ23441 1. Spacer groups with the 4 adjacent residues from neighboringepitopes were utilizied as query sequences. 2. BLAST search parameters:Expect 20000, Word size 2, Matrix PAM30, No. of alignments 250. 3.Resultant sequence matches with the lowest E value are presented first.●No sequence identified (except human Hepatitis B viral capsid) had an Evalue <1. 4. Asterisks (*) denote non-matching residues; plus signs (+)denote residues of similar chemical composition.

In conclusion, the degree of homology of the vaccine junctional regionsto human sequences was not dissimilar from other sequences, such as theHBV epitopes themselves which are regarded as non-self by the immunesystem and which are not associated with autoimmune manifestations.

E. Effective Optimization of Antigen Processing and Epitope Presentation

To demonstrate that optimized epigene constructs were efficient fordelivery of epitopes to the immune system, human lymphoblastoid celllines were transfected with the vaccine construct and which were thenused as APC in antigenicity assays (Livingston, B. D. et al., Vaccine.19:4652-4660 (2001)). These assays provide a means to determine therelative amounts of epitopes produced as a result of processing.Utilizing these assays, Livingston demonstrated that spacer optimizationcan enhance, by as much as a thousand fold, the yield of specificepitopes (Livingston, B. D. et al., Vaccine. 19:4652-4660 (2001)).Transfectants are generated with various vaccine constructs, includingGCR-5835 (see below), as constructs based on whole HBV genes. Theseresults demonstrate that the approach of processing optimization, basedon specific spacer residues, is highly effective. Furthermore, since thecell lines used for transfection are of human origin, these data providean important validation, in a human system, of the results describedabove obtained in HLA-transgenic mice.

4. Configuration, Formulation and Delivery of the Vaccine VaccineConfiguration

The HTL and CTL epigene constructs were designed and optimizedindependently. However, co-delivery of the HTL and CTL components in asingle DNA vaccine is considered optimal. Three vaccine alternativesinclude (1) the use of two separate CMV promoters; (2) the use of theCMV promoter in conjunction with an IRES, and (3) a construct encodingthe CTL+HTL components in a single reading frame (FIG. 29A). Forexamples of the third alternative, see Tables 18 and 19 (e.g. GCR-5835and GCR-3697). The immunogenicity of these different strategies wasevaluated utilizing HLA-A2 transgenic mice; the results are shown inFIG. 29B. It is apparent that each of the configurations inducedgenerally comparable CTL responses against all the HLA-A2 epitopes.Overall, the fused construct performed as well or better than the othervaccine configurations and it is currently regarded as the leadingvaccine configuration due to simplicity.

To create a vaccine better suited for human use and potentially augmentimmunogenicity, the nucleotide sequence of GCR-5835 (Table 18) wasmodified to match human codon frequencies, increase mRNA stability andreduce mRNA secondary structure. The immunogenicity of the modifiedconstruct, referred to as GCR-3697 (Table 19), was compared to that ofGCR-5835 in HLA-A2 transgenic mice. Animals were immunized i.m. witheither 5 μg or 50 μg of the human optimized GCR-3697 or GCR-5835 and CTLresponses were measured using primary IFN-γ ELISPOT assays (FIG. 30). Atthe 50 μg dose there was an average five-fold increase in the magnitudeof CTL responses to epitopes core 18, env 183 and pol 538 in the animalsimmunized with GCR-3697. The HTL responses induced by the two constructswere generally equivalent (data not shown). Since the GCR-3697 vaccineconstruct appeared to have greater potency, with respect to CTLimmunogenicity, and may possess attributes that will enhanceimmunogenicity in humans, its selection as the primary vaccine iswarranted.

TABLE 18 Epigene fusion construct in GCR-5835 plasmid GCR-5835Polynucleotide SEQ ID NO: 205

TCTCTGGGTTCCAGGATCACGGGGCTTCTTGCTTAGCTTGGGCATCCACCTAAATGCTGCTGCAAAATACACATCTTTTCCTTGGCTCCTTAATGCCGCCGCTAGGTTTTCATGGCTGAGTCTGCTAGTACCTTTCAATGCGGCTTTCCCACATTGCCTAGCTTTTAGCTATATGAAAGCTGCTTTAGTCGTGGACTTTTCACAGTTTAGCAGAGGAGCAATCCTGCTGCTATGTCTGATATTCCTTCTAAACGCAGCAGCCCACACACTCTGGAAAGCTGGTATCCTTTACAAGAAAGCCTGGATGATGTGGTATTGGGGACCCAGCCTCTACAAAGCATACCCTGCCCTGATGCCACTATACGCATGCATTGGCGCGGCAGCCTGGTTATCCCTTTTAGTACCGTTTGTCAACGCCGCAGCGGGATTTCTATTAACCAGAATCCTGACGATTAATGCTGCCGCCATTCCGATCCCAAGTTCCTGGGCATTCAAAGCAGCCGCGGCGTATCTGGTTTCATTTGGCCTATGGAACCTGCCAAGCGACTTCTTTCCTTCTGTTAAGGCCGCTGCTTTCCTCCCCTCCGATTTCTTTCCATCGGTGAAAGCCGCTGCCGACCTCCTTGATACCGCGAGCGCTCTGTACAACTCGTGGCCAAAATTCGCAGTTCCAAACCTAAAAGCCGCCGCCAGTGCCATTTGTTCCGTGGTAAGGAGAAAATTATCACTCGACGTGTCCGCAGCATTTTATAACGCTGCTGCAAAGTTTGTCGCACGATGGACATTGAAGGCTGCAGCGAAAGCAGCAAATGTATCAATACCCTGGACCCACAAGGGTGCAGCCGGGCTGTCTAGGTATGTGGCGAGGCTAAACGCCGCCGCCTCAACACTGCCTGAGACTACTGTCGTGAGACGCAAACACCCTGCCGCAATGCCCCACCTGCTGAAAGCAGCCGCACGATGGATGTGCCTCAGAAGATTCATAATAAACGCTTCTTTCTGTGGGTCACCCTACAAAGCCGCTTACATGGACGATGTGGTCCTCGGACTGAATGCCCTCTGGTTCCATATCAGCTGCCTGACATTCAAGGCAGCCGCCACCCCCGCTCGTGTGACAGGAGGTGTCTTCAAAGCCGCGGCACTGACTTTCGGTCGGGAAACTCTATTCCAATATAAGCAGGCCTTCACATTCTCCCCAACATACAAGAACGCAGGAACTTCTTTTGTGTATGTCCCTTCCGCTGTGAACCCAGCAGACGGACCCGGGCCTGGCCTGTGCCAGGTCTTCGCCGACGCAACTCCCACAGGGTGGGGGCTGGGGCCAGGACCAGGCAGGCACTACCTGCATACTCTGTGGAAGGCAGGAATCCTCTATAAAGGGCCCGGCCCAGGCCCTCACCACACCGCCCTGAGGCAGGCCATCCTGTGCTGGGGGGAGCTCATGACCCTGGCCGGACCTGGACCCGGGGAGAGCAGACTGGTGGTGGATTTCAGCCAATTCAGCAGAGGAAACGGACCCGGCCCTGGGCCTTTTCTGCTGGCTCAGTTTACATCTGCTATTTGTTCTGTGGTCGGCCCCGGGCCCGGACTCGTGCCTTTCGTGCAGTGGTTCGTGGGACTGTCCCCTACAGTCGGGCCCGGCCCAGGGCTGCATCTGTACTCCCACCCAATCATCCTCGGCTTCCGCAAGATTGGACCCGGCCCAGGCTCCAGCAATCTCTCCTGGCTCTCTCTGGACGTGTCTGCCGCCTTTGGCCCTGGACCAGGCCTGCAAAGCCTGACTAATCTGCTCAGCAGCAACCTGTCCTGGCTGGGACCTGGCCCAGGGGCTGGCTTCTTTCTGCTCACCCGGATTCTCACAATTCCCCAGTCCGGACCAGGACCAGGAGTCAGTTTCGGGGTGTGGATCAGGACCCCTCCTGCTTATAGACCACCCAATGCTCCAATCGGCCCCGGCCCTGGCGTCGGGCCACTGACCGTGAATGAGAAGCGCCGGCTGAAGCTCATCGGCCCTGGCCCTGGCAAGCAGTGCTTTCGCAAACTGCCCGTGAACAGACCTATTGATTGGGGCCCCGGCCCTGGAGCAGCCAACTGGATTCTCAGGGGAACAAGCTTCGTCTACGTGCCCGGGCCCGGACCAGGGAAGCAGGCTTTTACCTTCTCTCCCACTTACAAGGCCTTCCTCTGTGGGCCAGGCCCCGGCGCCAAGTTTGTGGCAGCATGGACCCTCAAAGCC

GCR-5835 Polypeptide SEQ ID NO: 206

FPHCLAFSYMKAALVVDFSQFSRGAILLLCLIFLLNAAAHTLWKAGILYKKAWMMWYWGPSLYKAYPALMPLYACIGAAAWLSLLVPFVNAAAGFLLTRILTINAAAIPIPSSWAFKAAAEYLVSFGVWNLPSDFFPSVKAAAFLPSDFFPSVKAAADLLDTASALYNSWPKFAVPNLKAAASAICSVVRRKLSLDVSAAFYNAAAKFVAAWTLKAAAKAANVSIPWTHKGAAGLSRYVARLNAAASTLPETTVVRRKHPAAMPHLLKAAARWMCLRRFIINASFCGSPYKAAYMDDVVLGVNALWFHISCLTFKAAATPARVTGGVFKAAALTFGRETVLEYKQAFTFSPTYKNAGTSFVYVPSALNPADGPGPGLCQVFADATPTGWGLGPGPGRHYLHTLWKAGILYKGPGPGPHHTALRQAILCWGELMTLAGPGPGESRLVVDFSQFSRGNGPGPGPFLLAQFTSAICSVVGPGPGLVPFVQWDVGLSPTVGPGPGLHLYSHPIILGFRKIGPGPGSSNLSWLSLDVSAAFGPGPGLQSLTNLLSSNLSWLGPGPGAGFFLLTRILTIPQSGPGPGVSFGVWIRTPPAYRPPNAPIGPGPGVGPLTVNEKRRLKLIGPGPGKQCFRKLPVNRPIDWGPGPGAANWILRGTSFVYVPGPGPGKQAFTFSPTYKAFLCGPGPGAK

TABLE 19 Epigene fusion construct in GCR-3697 plasmid GCR-3697Polynucleotide SEQ ID NO:207

AGCAGAGGCTTTCTCCTGTCCCTGGGCATCCACCTGAACGCCGCTGCAAAGTACACCAGCTTCCCCTGGCTGCTCAACGCCGCTGCCCGGTTCAGCTGGCTGTCCCTGCTCGTGCCCTTCAACGCAGCCTTCCCCCACTGCCTGGCCTTCAGCTACATGAAAGCAGCCCTGGTGGTCGACTTCTCCCAGTTCAGCCGGGGAGCCATCCTGCTCCTGTGCCTGATCTTTCTGCTCAACGCCGCTGCCCACACCCTGTGGAAGGCTGGCATCCTGTACAAGAAAGCCTGGATGATGTGGTACTGGGGACCCAGCCTGTACAAGGCATATCCAGCCCTGATGCCCCTGTACGCCTGCATCGGAGCTGCCGCATGGCTGAGCCTCCTGGTGCCCTTCGTGAACGCCGCTGCCGGGTTCCTGCTGACAAGAATCCTGACCATCAACGCCGCAGCCATTCCTATCCCCTCCAGCTGGGCCTTCAAGGCAGCCGCCGAGTACCTGGTGAGCTTCGGAGTCTGGAACCTGCCCAGCGACTTCTTTCCCAGCGTGAAAGCCGCAGCCTTCCTGCCCTCCGACTTCTTTCCCAGCGTGAAGGCCGCAGCCGATCTCCTGGACACCGCTAGCGCCCTGTACAACAGCTGGCCCAAGTTCGCCGTGCCCAACCTGAAGGCCGCAGCCAGCGCCATCTGCAGCGTGGTCAGACGGAAGCTGTCCCTCGATGTGAGCGCCGCTTTCTACAACGCCGCCGCAAAGTTCGTGGCCGCCTGGACCCTGAAAGCCGCTGCCAAGGCAGCCAACGTGAGCATCCCCTGGACCCACAAAGGAGCCGCAGGACTGAGCCGGTATGTGGCCAGACTGAACGCCGCTGCCAGCACCCTGCCCGAGACCACAGTGGTCAGACGGAAGCACCCCGCCGCCATGCCCCACCTGCTGAAGGCCGCAGCCCGGTGGATGTGCCTCAGACGGTTCATCATCAACGCTTCCTTCTGTGGCAGCCCCTACAAGGCCGCCTACATGGATGACGTGGTCCTGGGAGTGAACGCCCTCTGGTTCCACATCAGCTGCCTCACCTTCAAAGCCGCTGCCACACCCGCAAGAGTGACCGGAGGCGTGTTCAAGGCTGCAGCCCTGACCTTCGGCCGGGAGACCGTGCTGGAGTACAAGCAGGCCTTCACCTTCAGCCCCACCTACAAGAACGCCGGCACCAGCTTTGTGTACGTCCCAAGCGCCCTGAATCCCGCAGACGGCCCCGGCCCCGGACTGTGCCACGTGTTCGCCGATGCCACACCAACCGGATGGGGCCTGGGCCCTGGACCCGGCAGACACTACCTGCATACCCTGTGGAAGGCAGGAATCCTGTACAAAGGCCCCGGCCCTGGACCCCATCACACCGCTCTGCGGCAGGCCATCCTGTGCTGGGGCGAGCTCATGACTCTGGCAGGACCCGGCCCCGGCGAATCCAGGCTGGTGGTGGACTTTAGCCAGTTCTCCAGAGGCAACGGACCCGGCCCAGGACCCTTCCTGCTCGCCCAGTTCACCAGCGCCATCTGCAGCGTGGTCGGACCTGGCCCAGGACTGGTGCCCTTCGTGCAGTGGTTCGTCGGCCTCAGCCCCACCGTCGGACCTGGCCCCGGCCTGCACCTCTACAGCCACCCTATCATTCTGGGCTTCAGAAAGATCGGACCAGGCCCCGGCTCCAGCAACCTGTCCTGGCTCAGCCTGGACGTCAGCGCAGCCTTCGGACCCGGCCCTGGCCTGCAGAGCCTGACCAACCTGCTCAGCAGCAACCTCAGCTGGCTGGGCCCAGGACCCGGCGCAGGCTTCTTTCTGCTCACCAGAATCCTGACCATCCCTCAGAGCGGCCCCGGACCAGGCGTGAGCTTCGGCGTGTGGATTCGGACTCCTCCCGCCTACAGACCCCCAAATGCCCCCATCGGCCCAGGACCCGGCGTCGGACCTCTGACTGTGAACGAGAAGCGGAGACTGAAGCTGATCGGCCCCGGACCAGGCAAACAGTGCTTCAGGAAGCTCCCTGTGAACAGACCTATCGACTGGGGCCCCGGACCCGGCGCAGCCAACTGGATTCTGAGAGGCACCAGCTTCGTGTACGTCCCTGGACCCGGCCCTGGCAAGCAAGCCTTCACCTTCAGCCCCACCTACAA

GCR-3697 Polypeptide SEQ ID NO: 208

NAAFPHCLAFSYMKAALVVDFSQFSRGAILLLCLIFLLNAAAHTLWKAGILYKKAWMMWYWGPSLYKAYPALMPLYACIGAAAWLSLLVPFVNAAAGFLLTRILTINAAAIPIPSSWAFKAAAEYLVSFGVWNLPSDFFPSVKAAAFLPSDFFPSVKAAADLLDTASALYNSWPKFAVPNLKAAASAICSVVRRKLSLDVSAAFYNAAAKFVAAWTLKAAAKAANVSIPWTHKGAAGLSRYVARLNAAASTLPETTVVRRKHPAAMPHLLKAAARWMCLRRFIINASFCGSPYKAAYMDDVVLGVNALWFHISCLTFKAAATPARVTGGVFKAAALTFGRETVLEYKQAFTFSPTYKNAGTSFVYVPSALNPADGPGPGLCQVFADATPTGWGLGPGPGRHYLHTLWKAGILYKGPGPGPHHTALRQAILCWGELMTLAGPGPGESRLVVDFSQFSRGNGPGPGPFLLAQFTSAICSVVGPGPGLVPFVQWFVGLSPTVGPGPGLHLYSHPIILGFRKIGPGPGSSNLSWLSLDVSAAFGPGPGLQSLTNLLSSNLSWLGPGPGAGFFLLTRILTIPQSGPGPGVSFGVWIRTPPAYRPPNAPIGPGPGVGPLTVNEKRRLKLIGPGPGKQCGRKLPVNRPIDWGPGPGAA

Vaccine Formulation

Naked-DNA vaccines have not proved optimal for delivering vaccineimmunogens in humans (Wang, et al., Science 282:476 (1998)). Wetherefore selected an alternative formulation based on the use of apolymer surfactant, polyvinylpyrrolidone (PVP). This is a non-condensingdelivery system designed to increase the tissue distribution of the DNA,to protect DNA from degradation and to increase uptake by cells. PVP isa commonly used pharmaceutical formulation excipient that is nontoxicand approved for human clinical use. The properties and mechanisms ofaction for PVP appear to be very similar to the nonionic blockcopolymer, CRL1005. Safety, toxicity and biodistribution/clearance testswere completed to support use for a HIV-1 vaccine program. The data notonly support the safety of the formulation, but also cellular uptake ofDNA appears to be increased by more than a log, based on comparison tonaked DNA. Thus, the use of this delivery system can be supported byavailable data.

We evaluated the effects of the PVP on the immunogenicity of the CTL andHTL epitopes using an HIV-1 vaccine and several of the HBV vaccineconstructs. Data obtained using an HIV-1 epitope encoding epigeneconstruct demonstrated that PVP increased the immunogenicity of epitopesthat were only poorly immunogenic when delivered in a naked-DNA vaccine(data not shown). The immunogenicity of GCR-5835 was evaluated in thecontext of three different formulations, PVP, naked DNA, and cardiotoxin(CT) pre-priming. CT pretreatment is an experimental approach commonlyutilized in laboratory animals to enhance effectiveness of naked DNAinjections. CT destroys muscle fibers which then take up DNA as theyregenerate (Davis, H. L. et al., Mol. Genet. 2:1847-1851 (1993)). Theresults are shown in FIG. 31. While CT pretreatment was the mosteffective at priming high magnitude responses, this approach is notclinically applicable. The PVP-formulated DNA increased the magnitude ofresponses for two of the six epitopes measured when compared to nakedDNA, while the frequency of positive responses was higher for five ofsix epitopes. This data establishes that the PVP formulation increasesthe potency of the vaccine as compared to a naked DNA delivery.

Vaccine Route of Administration and Delivery

A PVP-formulated DNA plasmid vaccine can be delivered intramuscularly(i.m.). The i.m. route of administration is commonly used for DNAvaccines. In preliminary experiments, we utilized an HBV prototypeepigene construct, pMin1, to evaluate various DNA delivery routes (Table20). In these experiments, i.m. needle delivery was compared withneedleless delivery of PVP-formulated DNA via Biojector and ballisticdelivery of gold particle/DNA via PowderJect. Overall, the i.m. needledelivery performed as well or better than the other delivery methodstested although other delivery methods may be used.

Improvements to the Naked-DNA Vaccine Technology

Naked DNA vaccines have proven to be relatively poor immunogens innon-human primates and humans but studies completed thus far were basedon the delivery of intact genes encoding full-length proteins, orepitopes without spacer optimizations. Despite its relatively modesthuman immunogenicity, naked DNA immunization does appear to beremarkably effective in “priming” CTL responses (Ramshaw, J. A. andRamsay, A. J., Immunol. Today 21:163-165 (2000)).

Epigene construct design and addition of PVP are utilized to increaseDNA uptake. Epigene constructs may include a small plasmid DNA backboneand a small vaccine insert that can enhance cellular uptake of DNA,relative to larger clinically tested constructs.

TABLE 20 Comparison of route of DNA delivery for induction of CTLresponses Comparison of route of DNA delivery for induction of CTLresponses Immunogenicity (SU)¹ Delivery HBV core 18 HIV pol 476 HBV pol455 IM 1342.9 (1.8) 1133.3 (1.3) 879.5 (2.1) ID  740.1 (1.5) 0 0Biojector  44.7 (3.9)  103.2 (1.4)  44.8 (3.1) Immunogenicity (SFC/10⁶CD8 cells)² Delivery HBV core 18 HIV pol 476 HBV pol 455 HIV env 120 HBVpol 551 HBV env 335 IM 285.0 (17.4) 147.5 (19.8) 155.0 (15.0) 60.0(15.6) 485.0 (24.9) 68.3 (8.2) Gene Gun 287.5 (23.8) 146.7 (24.2)  25.8(7.5)  0.8 (5.5)  35.5 (7.6) 35.8 (16.2) ¹Immunogenicity of pMin1 inHLA-A2 using different routes of delivery. CTL responses were measuredusing an in situ ELISA assay (McKinney, D. M. et al., J. Immunol.Methods. 237: 105-117 (2000)). ²Immunogenicity of pMin1 in HLA-A2 usingneedle IM or Gene Gun immunization CTL responses were measured using aprimary IFN-γELISPOT

The heterologous prime-boost regimen, using a DNA vaccine first andeither proteins or viral vectors to boost responses, is currentlyconsidered to be the most immunogenic for genetic vaccines (Ramshaw, J.A. and Ramsay, A. J., Immunol. Today 21:163-165 (2000)). Heterologousprime:boost approaches can be utilized as a component of HBV vaccinedelivery.

5. Potency and Characterization of the Vaccine

A. Relevant Levels of Immunogenicity are Obtained in HLA-Transgenic Mice

The magnitude of responses obtained using the GCR-5835 vaccine wasevaluated in HLA-A2-transgenic mice and compared to responses inducedfollowing immunization with the experimental lipopeptide vaccineCY-1899. The lipopeptide vaccine was selected for this evaluationbecause the core18 epitope is present in both vaccine constructs andCY-1899 is known to elicit a potent CTL response in healthy humans(Livingston, B. D. et al., J. Immunol. 159:1383-1392 (1997)). Responsesinduced in the mice are shown in FIG. 32. Splenocytes from miceimmunized with the GCR-5835 construct produced IFN-γ responses to allsix HLA-A2-restricted epitopes encoded in the construct; measured usingan ELISPOT assay (FIG. 32A). A response to the core 18 epitope inCY-1899 was also observed, but the magnitude was considerably lower thanthe core 18 epitope response induced using the GCR-5835 vaccineconstruct. However, after a 6 day restimulation with peptide, the core18 responses induced by these two different format vaccines were verysimilar (FIG. 32B).

The magnitude of responses obtained for the other A2-restricted epitopeswas found to be comparable to those known to mediate clearance of HBVinfection. We observed primary ELISPOT responses ranging fromapproximately 100 SFC/10⁶ CD8+ cells (env 335) to greater than 300SFC/10⁶ CD8+ cells (env 183), well within the range of other responsesdetected in acute infections as detailed in Section 1A.

B. Quality of Responses

Clearance of HBV is mediated by a complex series of molecular events,including indirect, lymphokines-mediated effects, as well as directlysis of infected cells, especially the ones harboring integrated virus.IFN-γ production was measured in all experiments described thus far,which is relevant since this lymphokine is involved in clearance of HBVinfection (Chisari, F. V. and Ferrari, C. Annu. Rev. Immunol. 13:29-60(1995); Guidotti, L. G. et al., Immunity. 4:25-36 (1996)). DNAimmunization has been shown to induce CTL capable of lytic activity(Ishioka, G. Y. et al., J. Immunol. 162:3915-3925 (1999)).HLA-transgenic mice can be also be immunized with GCR-5835 and/orGCR-3697.

The immunological assay results presented were generally derived usingpooled preparations of splenocytes from 3-6 mice. Additional experimentswere performed to determine if responses against multiple epitopes wereinduced in individual animals. HLA-A2 transgenic mice were immunizedeither once or twice, at a one week interval, with GCR-5835 formulatedin PVP. Splenocytes from individual animals were harvested separatelyand restimulated with a pool of the six HLA-A2 epitope peptides encodedin the vaccine. IFN-γ secretion was then measured in response toindividual peptides using an ELISPOT assay. After a single immunization,all the mice responded to at least one epitope, average response ratewas 4.2±2.0 epitope/mouse (FIG. 33A). After a second immunization, theaverage number of epitopes recognized was increased to 5.6±0.5 (FIG.33B). These data have particular relevance in light of recent data onimmunodominance (Rodriguez, F. et al., J. Virol. 76:4251-4259 (2002)),and indicates that immunogen optimization and repeated immunizations maybe used to counterbalance the narrowness of responses resulting fromimmunodominance (Chen, M. et al., J. Virol. 74:7587-7599 (2000);Yewdell, J. W. et al., Annu. Rev. Immunol. 17:51-88 (1999)).

6. Summary and Conclusions

Multi-epitope CTL/HTL epigene constructs are effective for immunotherapyof chronic HBV infection and can be used in the treatment ofanti-viral-treated, chronically-infected individuals.

Processes used for identifying CTL and HTL epitopes suitable for use inthe design of vaccines are described above. The projected populationcoverage and immune response redundancy afforded by these epitope setsin different ethnic backgrounds is consistent with the breadth andmulti-specificity of responses naturally associated with resolution ofHBV infection. The vaccine design methods utilized to assemble themulti-epitope constructs entailed the optimization of proteosomalcleavage (CTL epitopes), and the minimization of junctional motifs (HTLepitopes).

Specific vaccine constructs were produced that induced potent CTLresponses in HLA-transgenic mice against most or all of the epitopesevaluated. The vaccine construct induces levels of HBV epitope-specificCTL in transgenic mice that are similar, in magnitude, to the responsesinduced using the CY-1899 vaccine, which is known to be immunogenic inhumans, and that are similar to the levels of CTL responses observed inhumans during resolution of HBV infection.

In addition, we showed how different vaccine configurations areeffective for simultaneous delivery of CTL and HTL epitopes. Epigeneconstructs may contain HTL and CTL epitopes that are co-linearlysynthesized from a single genetic insert and as such, the vaccine isreadily manufactured and stable.

A PVP-based DNA formulation is associated with increased activity, ascompared to naked DNA. Similarly, i.m. delivery appears to be, in thesystem investigated, the most practical and is associated with activityat least as good as other delivery methods (Biojector or gene gun). Acombination of priming with an optimized epigene construct formulated inPVP, followed by boosting with a viral vector can also be used.

TABLE 21 Codon Usage Table for Human Genes (Homo sapiens) Amino AcidCodon Number Frequency Phe UUU 326146 0.4525 Phe UUC 394680 0.5475 Total720826 Leu UUA 139249 0.0728 Leu UUG 242151 0.1266 Leu CUU 246206 0.1287Leu CUC 374262 0.1956 Leu CUA 133980 0.0700 Leu CUG 777077 0.4062 Total1912925 Ile AUU 303721 0.3554 Ile AUC 414483 0.4850 Ile AUA 1363990.1596 Total 854603 Met AUG 430946 1.0000 Total 430946 Val GUU 2104230.1773 Val GUC 282445 0.2380 Val GUA 134991 0.1137 Val GUG 559044 0.4710Total 1186903 Ser UCU 282407 0.1840 Ser UCC 336349 0.2191 Ser UCA 2259630.1472 Ser UCG 86761 0.0565 Ser AGU 230047 0.1499 Ser AGC 373362 0.2433Total 1534889 Pro CCU 333705 0.2834 Pro CCC 386462 0.3281 Pro CCA 3222200.2736 Pro CCG 135317 0.1149 Total 1177704 Thr ACU 247913 0.2419 Thr ACC371420 0.3624 Thr ACA 285655 0.2787 Thr ACG 120022 0.1171 Total 1025010Ala GCU 360146 0.2637 Ala GCC 551452 0.4037 Ala GCA 308034 0.2255 AlaGCG 146233 0.1071 Total 1365865 Tyr UAU 232240 0.4347 Tyr UAC 3019780.5653 Total 534218 His CAU 201389 0.4113 His CAC 288200 0.5887 Total489589 Gln CAA 227742 0.2541 Gln CAG 668391 0.7459 Total 896133 Asn AAU322271 0.4614 Asn AAC 376210 0.5386 Total 698481 Lys AAA 462660 0.4212Lys AAG 635755 0.5788 Total 1098415 Asp GAU 430744 0.4613 Asp GAC 5029400.5387 Total 933684 Glu GAA 561277 0.4161 Glu GAG 787712 0.5839 Total1348989 Cys UGU 190962 0.4468 Cys UGC 236400 0.5532 Total 427362 Trp UGG248083 1.0000 Total 248083 Arg CGU 90899 0.0830 Arg CGC 210931 0.1927Arg CGA 122555 0.1120 Arg CGG 228970 0.2092 Arg AGA 221221 0.2021 ArgAGG 220119 0.2011 Total 1094695 Gly GGU 209450 0.1632 Gly GGC 4413200.3438 Gly GGA 315726 0.2459 Gly GGG 317263 0.2471 Total 1283759 StopUAA 13963 Stop UAG 10631 Stop UGA 24607

TABLE 22 Codon Usage Table for Mouse Genes (Mus musculus) Amino AcidCodon Number Frequency Phe UUU 150467 0.4321 Phe UUC 197795 0.5679 Total348262 Leu UUA 55635 0.0625 Leu UUG 116210 0.1306 Leu CUU 114699 0.1289Leu CUC 179248 0.2015 Leu CUA 69237 0.0778 Leu CUG 354743 0.3987 Total889772 Ile AUU 137513 0.3367 Ile AUC 208533 0.5106 Ile AUA 62349 0.1527Total 408395 Met AUG 204546 1.0000 Total 204546 Val GUU 93754 0.1673 ValGUC 140762 0.2513 Val GUA 64417 0.1150 Val GUG 261308 0.4664 Total560241 Ser UCU 139576 0.1936 Ser UCC 160313 0.2224 Ser UCA 100524 0.1394Ser UCG 38632 0.0536 Ser AGU 108413 0.1504 Ser AGC 173518 0.2407 Total720976 Pro CCU 162613 0.3036 Pro CCC 164796 0.3077 Pro CCA 151091 0.2821Pro CCG 57032 0.1065 Total 535532 Thr ACU 119832 0.2472 Thr ACC 1724150.3556 Thr ACA 140420 0.2896 Thr ACG 52142 0.1076 Total 484809 Ala GCU178593 0.2905 Ala GCC 236018 0.3839 Ala GCA 139697 0.2272 Ala GCG 604440.0983 Total 614752 Tyr UAU 108556 0.4219 Tyr UAC 148772 0.5781 Total257328 His CAU 88786 0.3973 His CAC 134705 0.6027 Total 223491 Gln CAA101783 0.2520 Gln CAG 302064 0.7480 Total 403847 Asn AAU 138868 0.4254Asn AAC 187541 0.5746 Total 326409 Lys AAA 188707 0.3839 Lys AAG 3027990.6161 Total 491506 Asp GAU 189372 0.4414 Asp GAC 239670 0.5586 Total429042 Glu GAA 235842 0.4015 Glu GAG 351582 0.5985 Total 587424 Cys UGU97385 0.4716 Cys UGC 109130 0.5284 Total 206515 Trp UGG 112588 1.0000Total 112588 Arg CGU 41703 0.0863 Arg CGC 86351 0.1787 Arg CGA 589280.1220 Arg CGG 92277 0.1910 Arg AGA 101029 0.2091 Arg AGG 102859 0.2129Total 483147 Gly GGU 103673 0.1750 Gly GGC 198604 0.3352 Gly GGA 1514970.2557 Gly GGG 138700 0.2341 Total 592474 Stop UAA 5499 Stop UAG 4661Stop UGA 10356

Example 18 Proteasomal Processing of a Hepatitis B Virus PolyepitopeGene Product In Vitro

Introduction

A CTL epitope-based approach to the design of a vaccine against chronichepatitis B virus (HBV) was taken. A synthetic gene encoding a series of16 epitopes was made where the epitopes are separated by amino acidspacers designed to enhance proteolytic processing. In vitro translationas well as transient expression of this HBV polyepitope minigene in ahuman cell line results in rapid degradation of the polyprotein, asexpected for a gene product that is labile to proteasome activity. ThisHBV polyepitope (AOSIb) was fused directly to a fluorescent protein forease of detection. Addition of proteasome-specific inhibitors totransfected cultures showed a marked increase in the amount of fusionprotein present in cells, as judged by FACS analysis, fluorescencemicroscopy and Western blot. The ability of proteasome inhibitors toblock processing of the polyepitope gene product, combined with in vivoimmunogenicity to the pathogen-specific epitopes in the DNA plasmid showthat the amino acid spacers were efficacious in assuring class Iprocessing. A subsequent HBV polyepitope construct (AOSIb.2) was madethat incorporates several amino acid additions expected to improveproteasomal processing. The results show that the spacer sequences usedin this HBV polyepitope plasmid can promote proteasome processing of theexpressed polypeptide and efficient CTL epitope presentation.

2. Experimental Approach

DNA expression cassettes were designed where HBV polyepitope stringswere fused to a fluorescent marker to facilitate protein detection andquantitation in vitro. Spacers of varying composition were added to oneconstruct to evaluate potential improvements in intracellular epitopeprocessing. Proteasome inhibitors were added to plasmid-transfectedcells to prevent proteasome degradation of cytosolic proteins. Thepresence of fusion proteins was monitored by fluorescent markerdetection via FACS, fluorescence microscopy or Western blots. The amountof fluorescence trapped in the cells was quantified to look for changesin polyprotein processing. The effect on in vivo immunogenicity inHLA-A2 transgenic mice was also measured for both plasmids to determineif the amino acid spacers had beneficial effects.

3. Composition of HBV Polyepitope Constructs

HBV AOSIb and HBV AOSIb2 carry virus specific epitopes that areoptimized. The constructs encode HLA-A2, HLA-A3 and HLA-B7 supertypeepitopes, 16 epitopes total. The HBV AOSIb2 construct has additionalamino acids added to enhance proteasomal processing while the HBV AOSIbhas no added residues. A schematic and the amino acid sequence of theCTL constructs HBV AOSIb and HBV AOSIb2 are shown in FIG. 34 and Tables23-24. An example of a polynucleotide sequence encoding HBV AOSIb andHBV AOSIb2 is shown in Tables 23-24.

TABLE 23 Epigene encoded by HBV AOSIb construct HBV AOSIb PolynucleotideSEQ ID NO :209

GGGTCCAGAGGACACACCCTGTGGAAGGCCGGAATCCTGTATAAGGCCAAGTTCGTGGCTGCCTGGACCCTGAAGGCTGCCGCTTTCCTGCCTAGCGATTTCTTTCCTAGCGTGTTCCTGCTGTCCCTGGGAATCCACCTGTATATGGATGACGTGGTGCTGGGAGTGGGACTGTCCAGGTACGTGGCTAGGCTGTTCTTGCTGACCAGAATCCTGACCATCTCCACCCTGCCA GAGACCACCGTGGTGAGGAGGCAGGCCTTCACCTTTAGCCCTACCTATAAGTGGCTGAGCCTGCTGGTGCCCTTTGTGATCOCTATCCCTAGCTCCTGGGCTTTCACCCCAGCCAGGGTGACCGGAGGAGTGTTTAAGGTGGGAAACTTCACCGGCCTGTATCTGCCCAGCGATTTCTTTCCTAGCGTGACCCTGTGGAAGGCCGGGATCCTGTACAAGAATGTGTCCATCCCTTGGACCCACAAGCTGGTGGTGGACTTTTCCCAGTTCAGCAGATCCGCTATCTGC

AOSIb Polypeptide SEQ ID NO: 210

TABLE 24 Epigene encoded by HBV AOSIb2 construct HBV AOSIb2Polynucleotide SEQ ID NO: 211

GGGTCCAGAGGACACACCCTGTGGAAGGCCGGAATCCTGTATAAGGCCAAGTTCGTGGCTGCCTGGACCCTGAAGGCTGCCGCTTTCCTGCCTAGCGATTTCTTTCCTAGCGTGAACTTCCTGCTGTCCCTGGGAATCCACCTGTATATGGATGACGTGGTGCTGGGAGTGGGACTGTCCAGGTACGTGGCTAGGCTGTTCCTGCTGACCAGAATCCTGACCATCTCCACCCTGCCAGAGACCACCGTGGTGAGGAGGCAGGCCTTCACCTTTAGCCCTACCTATAAGGGAGCCGCTGCCTGGCTGACGCTGCTGGTGCCCTTTGTGAATATCCCTATCCCTACGTCCTGGGCTTTCAAGACCCCAGCCAGGGTGACCGGAGGAGTGTTTAAGGTGGGAAACTTCACCGGCCTGTATAACCTGCCCAGCGATTTCTTTCCTAGCGTGAAGACCCTGTGGAAGGCCGGAATCCTGTACAAGAATGTGTCCATCCCTTGGACCCACAAGGGAGCCGCTCTGGTGGTGGACTTTTCCCAGTTCAGCAGAAATTCCGCTATCTGCTCCGTGGTGAGGAGAGCTCTGATGCCACTGT

HBV AOSIb2 Polypeptide SEQ ID NO: 212

4. In Vitro Protein Expression and Detection Method

Transient transfection of human 293 cell lines was carried out withplasmids encoding the fluorescent-conjugated polyepitopes HBV AOSIb orHBV AOSIb.2, or the fluoresence reporter plasmid with no epitopes. 5 μMof the irreversible proteasome inhibitor MG132 was added 24 hourspost-transfection. Fluorescence was detected in live cells by eitherflow cytometry (FACS) or fluoresecence microscopy within 24 hours of theaddition of the proteasome inhibitor. The increase in the number offluorescent cells transfected with plasmid HBV AOSIb in the presence orabsence of proteasome inhibitor was measured as shown in FIG. 35A, as afunction of incubation time with the inhibitor. FIG. 38 shows thecomparison in number of fluorescent cells detected (by FACS) after 24hour incubation with inhibitor for cells transfected with the threedifferent plasmids. The more profound effect was noted for thespacer-optimized plasmid HBV AOSIb.2. The number of cells expressing thevarious fluorescent fusion proteins was also measured by fluorescencemicroscopy of live cells as shown in FIG. 39. Western blot detection wasperformed by preparing whole cell lysates from transfected cells,separating proteins by gel electrophoresis, transferring to blottingmembranes, and detecting proteins with an antibody against the fusionpartner protein. The increase in amount of proteins detectable uponaddition of the proteasome inhibitors lactacystin (25 μM) or MG132 (5μM) was then determined. The results are shown in FIG. 36.

5. Mouse Immunogenicity Assay Method

Transgenic HLA-A2 mice were injected i.m. with 100 ug of a plasmidencoding HBV AOSIb or HBV AOSIb2 polyepitopes. Mice were sacrificed 14days later and their spleens were homogenized to collect T lymphocytesand APCs. Cells were stimulated in culture with peptides correspondingto the various HBV epitopes. The secretion of IFN-γ was measured by amodified ELISA method (to detect secretory units). The results aresummarized in Table 25.

TABLE 25 HLA-A2 Tg mice immunogenicity for plasmids AOSIb and AOSIb.2HBV HBV AOSIb_((100 ug dose)) AOSIb2_((100 ug dose)) Epitope magnitudefrequency magnitude frequency core 18  102.7_((1.8)) 6/6  480.6_((1.4))6/6 pol 562 — 0/6  260.2_((2.1)) 6/6 pol 538 2643.8_((1.3)) 6/62332.3_((1.4)) 6/6 pol 455 2234.6_((1.3)) 6/6  334.3_((1.3)) 6/6 env 183 877.5_((1.3)) 6/6  962.8_((1.3)) 6/6 env 335   6.1 1/6  44.9_((1.6))6/6 pol 642 1859.8_((1.6)) 6/6 1819.0_((1.6)) 6/66. Summary of Results

The HBV DNA constructs carry virus specific epitopes in optimizedcassettes able to elicit CTL responses, and additional amino acids wereintroduced between the epitopes of one construct to potentially enhanceproteasomal processing and thereby class I presentation of antigen.

Both HBV-fluorescent protein fusions were more labile than thefluorescent protein alone, suggesting the HBV polyepitopes are readilydegraded and drive the degradation of the whole fusion product.Proteasome inhibitors allow the detection of greater amounts offluorescent fusion products but have no effect on the fusion partner ifexpressesed alone, indicating that this is indeed a cytosomal proteasomeactivity enhanced by the polyepitopes. The effect of proteasomeinhibitor is more pronounced for the spacer-optimized HBV AOSIb2 productthan for the HBV AOSIb fusion protein indicating that the processingsites added to the HBV AOSIb2 molecule had the desired effect ofincreasing its processivity. Studies in HLA-A2 transgenic mice showed animprovement in immunogenicity of several epitopes for the “optimized”HBV AOSIb2 plasmid compared to HBV AOSIb.

Example 19 Epitope-Specific T Cell Responses Measured in HLA TransgenicMice Immunized with GCR-3697

Epitope-specific T cell responses were measured in HLA transgenic miceimmunized with GCR-3697 using splenic lymphocytes obtained 11-14 daysfollowing immunization (FIG. 37). Groups of 6-9 HLA-transgenic mice wereimmunized bilaterally with 100 μg of DNA in the tibialis anteriormuscle. DNA was delivered in either PBS or PVP formulations; in the caseof PBS formulations the injection site was pre-treated by cardiotoxininjection. Mice immunized with PVP based formulations were immunizedtwice with 100 μg of DNA in a four day period.

CTL responses were measured using an in situ ELISA assay based on theproduction of IFN-γ. Assays were conducted by culturing splenocytes(2.5×10⁷) with peptide (1 μg/ml) and irradiated lipopolysaccharide(LPS)-activated splenocytes (10⁷) in RPMI medium for 6 days at 37° C. in5% CO₂. After the 6-day stimulation, serially diluted splenocytes werecultured for 20 hours, with and without peptide (1 μg/ml), and 10⁵HLA-matched Jurkat target cells. Assays were performed on ELISA plates(Costar, Corning, N.Y.) pre-coated with rat monoclonal antibody specificfor murine IFN-γ (Clone RA-6A2, BD Biosciences/Pharmingen). Thefollowing day, the cells were removed by washing the plates with PBSwith 0.05% Tween-20 and the amount of IFN-γ secreted was measured usinga sandwich format ELISA. A biotinylated rat monoclonal antibody (cloneXMG1.2, BD Biosciences/Pharmingen) was used to detect captured IFN-γ.Horseradish peroxidase-coupled strepavidin (Zymed) and 3,3′,5,5′tetramethylbenzidine and H₂O₂ (ImmunoPure TMB Substrate Kit, Pierce)were used according to the manufacturer's directions for colordevelopment. The absorbance was read at 450 nm on a Labsystems MultiskanRC ELISA plate reader. In situ ELISA data was converted to secretoryunits (SU) for evaluation (McKinney et al 2000).

Overall, GCR-3697 induced CTL responses to multiple epitopes restrictedby a variety of HLA-supertypes. The magnitude and the breadth of theresponses induced are consistent with the nature of the immune responsesgenerally considered to of therapeutic value.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books,entries in sequence databases, or other disclosures) in the Background,Definitions, Detailed Description, and Examples is hereby incorporatedherein by reference.

Those skilled in the art will know, or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments of the invention described herein. These equivalents areintended to be encompassed by the following claims.

1. l A polynucleotide comprising a nucleic acid sequence selected fromthe group consisting of: nucleotides +1 to 1248 of SEQ ID NO: 71,nucleotides +1 to 2292 of SEQ ID NO: 205, and nucleotides +1 to 2232 ofSEQ ID NO:
 207. 2. A composition comprising the polynucleotide ofclaim
 1. 3. An isolated cell comprising the polynucleotide of claim 1.4. A method of inducing an immune response against hepatitis B virus(HBV) in an individual in need thereof, comprising administering thepolynucleotide of claim 1, the composition of claim 2, or the cell ofclaim 3 to said individual.